CA IX-specific inhibitors

ABSTRACT

Therapeutic methods for inhibiting the growth of preneoplastic/neoplastic vertebrate cells that abnormally express MN protein are disclosed. Screening assays are provided for identifying compounds, preferably organic compounds, preferably aromatic and heterocylic sulfonamides, which inhibit the enzymatic activity of MN/CA IX and that are useful for treating patients with preneoplastic/neoplastic disease. Further, the CA IX-specific inhibitors when labeled can also be used diagnostically/prognostically for preneoplastic/neoplastic disease, and for imaging use, for example, to detect hypoxic precancerous cells, tumors and/or metastases, by selectively binding to activated CA IX, preferably CA IX activated under hypoxic conditions, and not to inactive CA IX. Such detection of hypoxic conditions can be helpful in determining effective treatment options, and in predicting treatment outcome and the prognosis of disease development.

This application is a continuation of U.S. application Ser. No.11/222,986 (filed Sep. 8, 2005), now U.S. Pat. No. 7,833,734, which is acontinuation-in-part of U.S. Ser. No. 10/723,795 (filed on Nov. 26,2003), now U.S. Pat. No. 7,550,424, and this application claims priorityfrom now abandoned U.S. Provisional Application No. 60/609,103 (filed onSep. 9, 2004). Said U.S. Ser. No. 10/723,795 claims priority from U.S.Provisional Application Nos. 60/429,089 (filed on Nov. 26, 2002),60/489,473 (filed on Jul. 22, 2003), and 60/515,104 (filed on Oct. 28,2003). This application hereby declares priority under 35 USC §120 fromthe above-identified applications. The above priority applications andparent U.S. application Ser. No. 11/222,986 are hereby incorporated byreference.

REFERENCE TO SEQUENCE LISTING

The Sequence Listing, filed electronically and identified asUSSN-11-929652-SEQ-LISTING, was created on Feb. 26, 2008, is 31.6 kb insize and is hereby incorporated by reference.

FIELD OF THE INVENTION

The present invention is in the general area of medical genetics and inthe fields of chemistry, biochemical engineering, and oncology. Morespecifically, it relates to the use of organic and inorganic compounds,preferably aromatic and heterocyclic sulfonamides, to treatpreneoplastic and/or neoplastic diseases by specifically inhibiting thecarbonic anhydrase activity of the oncoprotein now known alternativelyas the MN protein, the MN/CA IX isoenzyme, the MN/G250 protein or simplyMN/CA IX or CA IX or MN. The present invention also relates to methodsof treating preneoplastic and/or neoplastic diseases characterized byMN/CA IX overexpression by administering cell membrane-impermeant,inhibitors of MN/CA IX, preferably pyridinium derivatives of aromaticand heterocyclic sulfonamides. The invention further concernsdiagnostic/prognostic methods including imaging methods, forpreneoplastic/neoplastic diseases, using the disclosed potent CAIX-specific inhibitors, and gene therapy with vectors conjugated to saidinhibitors.

BACKGROUND OF THE INVENTION

The instant inventors, Dr. Silvia Pastorekova and Dr. Jaromir Pastorek,with Dr. Jan Zavada [“Zavada et al.”], discovered MN/CA IX, a cancerrelated cell surface protein originally named MN. [73, 123; Zavada etal., U.S. Pat. No. 5,387,676 (Feb. 7, 1995).] Zavada et al., WO 93/18152(published 16 Sep. 1993) and Zavada et al., WO 95/34650 (published 21Dec. 1995) disclosed the discovery of the MN gene and protein and thestrong association of MN gene expression and tumorigenicity led to thecreation of methods that are both diagnostic/prognostic and therapeuticfor cancer and precancerous conditions. Zavada et al. disclosed furtheraspects of the MN/CA IX protein and the MN/CA9 gene in Zavada et al., WO00/24913 (published 4 May 2000).

Zavada et al. cloned and sequenced the MN cDNA and gene, and revealedthat MN belongs to a carbonic anhydrase family of enzymes that catalyzethe reversible hydration of carbon dioxide to bicarbonate and proton[66, 72]. MN protein (renamed to carbonic anhydrase IX, CA IX) iscomposed of an extracellular part containing a N-terminalproteoglycan-like region and a catalytically active carbonic anhydrasedomain. It is anchored in the plasma membrane by a single transmembraneregion and a short intracytoplasmic tail.

Expression of CA IX is restricted to only few normal tissues [74], butis tightly associated with tumors [123]. It is also regulated by celldensity in vitro [52] and is strongly induced by tumor hypoxia both invitro and in vivo [121]. Numerous clinical papers describe the value ofCA IX as an indicator of poor prognosis. All CA IX-related studiesperformed so far support the assumption made in the original Zavada etal., U.S. Pat. No. 5,387,676 that CA IX is useful as a diagnostic and/orprognostic tumor marker and as a therapeutic target.

MN/CA IX consists of an N-terminal proteoglycan-like domain that isunique among the CAs, a highly active CA catalytic domain, a singletransmembrane region and a short intracytoplasmic tail [66, 72, 74,116]. CA IX is particularly interesting for its ectopic expression in amultitude of carcinomas derived from cervix uteri, ovarian, kidney,lung, esophagus, breast, colon, endometrial, bladder, colorectal,prostate, among many other human carcinomas, contrasting with itsrestricted expression in normal tissues, namely in the epithelia of thegastrointestinal tract [8, 11, 21, 35, 41, 48, 50, 51, 56, 66, 72, 74,86, 110, 111, 113, 116, 121, 122].

Uemura et al. [112] reported in 1997 that the G250 antigen was identicalto MN/CA IX, years after MN/CA IX had been discovered and sequenced byZavada et al. {[73, 123]; see also Pastorek et al. [72] and Opavsky etal. [66]}. Uemura et al. [112] stated: “Sequence analysis and databasesearching revealed that G250 antigen is identical to MN a humantumor-associated antigen identified in cervical carcinoma (Pastorek etal., 1994).”

MN/CA 9 and MN/CA IX—Sequence Similarities

FIG. 1A-C shows the full-length MN/CA9 cDNA sequence of 1522 base pairs(bps) [SEQ ID NO: 1], and the full-length MN/CA IX amino acid (aa)sequence of 459 aa [SEQ ID NO: 2]. FIG. 2A-F provides the 10,898 bpgenomic sequence of MN/CA9 [SEQ ID NO: 3].

Computer analysis of the MN cDNA sequence was carried out using DNASISand PROSIS (Pharmacia Software packages). GenBank, EMBL, ProteinIdentification Resource and SWISS-PROT databases were searched for allpossible sequence similarities. In addition, a search for proteinssharing sequence similarities with MN was performed in the MIPS databankwith the FastA program [75].

The proteoglycan-like domain [aa 53-111; SEQ ID NO: 4] which is betweenthe signal peptide and the CA domain, shows significant homology (38%identity and 44% positively) with a keratan sulphate attachment domainof a human large aggregating proteoglycan aggrecan [28].

The CA domain [aa 135-391; SEQ ID NO: 5] is spread over 265 aa and shows38.9% amino acid identity with the human CA VI isoenzyme [5]. Thehomology between MN/CA IX and other isoenzymes is as follows: 35.2% withCA II in a 261 aa overlap [63], 31.8% with CA I in a 261 aa overlap [7],31.6% with CA IV in a 266 aa overlap [65], and 30.5% with CA III in a259 aa overlap [55].

In addition to the CA domain, MN/CA IX has acquired both N-terminal andC-terminal extensions that are unrelated to the other CA isoenzymes. Theamino acid sequence of the C-terminal part, consisting of thetransmembrane anchor and the intracytoplasmic tail, shows no significanthomology to any known protein sequence.

The MN gene (MN/CA9 or CA9) was clearly found to be a novel sequencederived from the human genome. The overall sequence homology between thecDNA MN/CA9 sequence and cDNA sequences encoding different CA isoenzymesis in a homology range of 48-50% which is considered by ones in the artto be low. Therefore, the MN/CA9 cDNA sequence is not closely related toany CA cDNA sequences.

Very few normal tissues have been found to express MN protein to anysignificant degree. Those MN-expressing normal tissues include the humangastric mucosa and gallbladder epithelium, and some other normal tissuesof the alimentary tract. Paradoxically, MN gene expression has beenfound to be lost or reduced in carcinomas and otherpreneoplastic/neoplastic diseases in some tissues that normally expressMN, e.g., gastric mucosa.

CA IX, Hypoxia and Acidification of Extracellular Environment

Strong association between CA IX expression and intratumoral hypoxia(either measured by microelectrodes, or detected by incorporation of ahypoxic marker pimonidazole, or by evaluation of extent of necrosis) hasbeen demonstrated in the cervical, breast, head and neck, bladder andnon-small cell lung carcinomas (NSCLC) [8, 11, 21, 35, 48, 56, 111,122]. Moreover, in NSCLC and breast carcinomas, correlation between CAIX and a constellation of proteins involved in angiogenesis, apoptosisinhibition and cell-cell adhesion disruption has been observed, possiblycontributing to strong relationship of this enzyme to a poor clinicaloutcome [8]. Hypoxia is linked with acidification of extracellularmilieu that facilitates tumor invasion and CA IX is believed to play arole in this process via its catalytic activity [86]. Thus, inhibitionof MN/CA IX by specific inhibitors is considered to constitute a novelapproach to the treatment of cancers in which CA IX is expressed.

Acidic extracellular pH (pHe) has been associated with tumor progressionvia multiple effects including up-regulation of angiogenic factors andproteases, increased invasion, and impaired immune functions [86, 124,125, 130, 132]. In addition, it can influence the uptake of anticancerdrugs and modulate the response of tumor cells to conventional therapy[86, 126]. Acidification of the tumor microenvironment was generallyassigned to accumulation of lactic acid excessively produced byglycolysis and poorly removed by inadequate tumor vasculature. A highrate of glycolysis is especially important for hypoxic cells thatlargely depend on anaerobic metabolism for energy generation. However,experiments with glycolysis-deficient cells indicate that production oflactic acid is not the only mechanism leading to tumor acidity. Thedeficient cells produce only diminished amounts of lactic acid, but formacidic tumors in vivo [134, 144]. A comparison of the metabolic profilesof the glycolysis-impaired and parental cells revealed that CO₂, inaddition to lactic acid, is a significant source of acidity in tumors[127]. That data indicates that carbonic anhydrases could contribute tothe acidification of the tumor microenvironment.

The CA IX isoform is identified herein as the best candidate for therole in acidifying the tumor microenvironment. First, CA IX is anintegral plasma membrane protein with an extracellularly exposed enzymeactive site [66, 72]. Second, CA IX has a very high catalytic activitywith the highest proton transfer rate among the known CAs [116]. Third,CA IX is present in few normal tissues, but its ectopic expression isstrongly associated with many frequently occurring tumors. Finally, CAIX level dramatically increases in response to hypoxia via a directtranscriptional activation of CA9 gene by HIF-1 [121], and itsexpression in tumors is a sign of poor prognosis [136]. Taken together,CA IX is herein considered to have all the qualities necessary tocontrol tumor pH. That concept is supported by the proof provided hereinthat CA IX has the capacity to acidify extracellular pH.

CAIs

Teicher et al. [106] reported that acetazolamide—the prototypical CAinhibitor (CAI)— functions as a modulator in anticancer therapies, incombination with different cytotoxic agents, such as alkylating agents;nucleoside analogs; platinum derivatives, among other such agents, tosuppress tumor metastasis and to reduce the invasive capacity of severalrenal carcinoma cell lines (Caki-1, Caki-2, ACHN, and A-498). Suchstudies demonstrate that CAIs may be used in the management of tumorsthat overexpress one or more CA isozymes. It was hypothesized that theanticancer effects of acetazolamide (alone or in combination with suchdrugs) might be due to the acidification of the intratumoral environmentensuing after CA inhibition, although other mechanisms of action of thisdrug were not excluded [20]. Chegwidden et al. 2001 hypothesized thatthe in vitro inhibition of growth in cell cultures, of human lymphomacells with two other potent, clinically used sulfonamide CAIs,methazolamide and ethoxzolamide, is probably due to a reduced provisionof bicarbonate for nucleotide synthesis (HCO₃ ⁻ is the substrate ofcarbamoyl phosphate synthetase II) as a consequence of CA inhibition[20].

All the six classical CAIs (acetazolamide, methazolamide, ethoxzolamide,dichlorophenamide, dorzolamide, and dichlorophenamide) used in clinicalmedicine or as diagnostic tools, show some tumor growth inhibitoryproperties [18, 78, 101, 102].

The inventors, Dr. Claudia Supuran and Dr. Andrea Scozzafava, reportedthe design and in vitro antitumor activity of several classes ofsulfonamide CAIs, shown to act as nanomolar inhibitors against theclassical isozymes known to possess critical physiological roles, suchas CA I, CA II and CA IV. Those compounds were also shown to exertpotent inhibition of cell growth in several leukemia, non-small celllung, ovarian, melanoma, colon, CNS, renal, prostate and breast cancercell lines, with GI₅₀ values of 10-75 nM in some cases [77, 91, 92,100].

Wingo et al. reported that three classic sulfonamide drugs(acetozolamide, ethoxzolamide and methoxzolamide) inhibited CA IXcarbonic anhydrase activity with values of K_(I) in the nanomolar range[116]. However, until the present invention, no systematicstructure-activity relationship study of sulfonamide inhibition of CAIX, alone or in comparison to other CA isozymes had been performed.

Certain pyridinium derivatives of aromatic/heterocyclic sulfonamideshave shown nanomolar affinities both for CA II, as well as CA IV, andmore importantly, they were unable to cross the plasma membranes in vivo[17].

Sterling et al. [85] investigated the functional and physicalrelationship between the downregulated in adenoma bicarbonatetransporter and CA II, by using membrane-impermeant sulfonamideinhibitors (in addition to the classical inhibitors such asacetazolamide), which could clearly discriminate between thecontribution of the cytosolic and membrane-associated isozymes in thesephysiological processes.

CAS

Carbonic anhydrases (CAs) form a large family of genes encoding zincmetalloenzymes of great physiological importance. As catalysts ofreversible hydration of carbon dioxide, these enzymes participate in avariety of biological processes, including respiration, calcification,acid-base balance, bone resorption, formation of aqueous humor,cerebrospinal fluid, saliva and gastric acid [reviewed in Dodgson et al.(27)]. CAs are widely distributed in different living organisms. Inhigher vertebrates, including humans, 14 different CA isozymes orCA-related proteins (CARP) have been described, with very differentsubcellular localization and tissue distribution [40, 93, 95, 94, 102].Basically, there are several cytosolic forms (CA 1-III, CA VII), fourmembrane-bound isozymes (CA IV, CA IX, CA XII and CA XIV), onemitochondrial form (CA V) as well as a secreted CA isozyme, CA VI [40,93, 94, 95, 102].

It has been shown that some tumor cells predominantly express only somemembrane-associated CA isozymes, such as CA IX and CA XII [2, 67, 68,78, 87, 93, 95]. Occasionally, nuclear localization of some isoenzymeshas been noted [64, 69, 70]. Not much is presently known about thecellular localization of the other isozymes.

CAs and CA-related proteins show extensive diversity in their tissuedistribution, levels, and putative or established biological functions[105]. Some of the CAs are expressed in almost all tissues (CA II),while the expression of others appears to be more restricted (e.g., CAVI and CA VII in salivary glands [32, 69, 71]. The CAs and CA-relatedproteins also differ in kinetic properties and susceptibility toinhibitors [82].

Most of the clinically used sulfonamides mentioned above aresystemically acting inhibitors showing several undesired side effectsdue to inhibition of many of the different CA isozymes present in thetarget tissue/organ (14 isoforms are presently known in humans) [93, 94,95, 102]. Therefore, many attempts to design and synthesize newsulfonamides were recently reported, in order to avoid such side effects[13, 17, 42, 62, 80, 99, 100]. At least four CA isozymes (CA IV, CA IX,CA XII and CA XIV) are associated to cell membranes, with the enzymeactive site generally oriented extracellularly [93, 94, 95, 102]. Someof these isozymes were shown to play pivotal physiological roles (suchas for example CA IV and XII in the eye, lungs and kidneys, CA IX in thegastric mucosa and many tumor cells) [3, 18, 22, 29, 49, 67, 68, 83, 93,94, 95, 102], whereas the function of other such isozymes (CA XIV) isfor the moment less well understood [93, 95]. Due to the extracellularlocation of these isozymes, if membrane-impermeant CA inhibitors (CAIs)could be designed, only membrane-associated CAs would be affected.

The first approach towards introducing the membrane-impermeability toCAIs from the historical point of view was that of attachingaromatic/heterocyclic sulfonamides to polymers, such aspolyethyleneglycol, aminoethyldextran, or dextran [39, 60, 107]. Suchcompounds, possessing molecular weights in the range of 3.5-99 kDa,prepared in that way, showed indeed membrane-impermeability due to theirhigh molecular weights, and selectively inhibited in vivo only CA IV andnot the cytosolic isozymes (primarily CA II), being used in severalrenal and pulmonary physiological studies [39, 60, 107]. Due to theirmacromolecular nature, such inhibitors could not be developed asdrugs/diagnostic tools, since in vivo they induced potent allergicreactions [39, 60, 93, 95, 107]. A second approach for achievingmembrane-impermeability is that of using highly polar, salt-likecompounds. Only one such sulfonamide has until recently been used inphysiological studies, QAS (quaternary ammonium sulphanilamide), whichhas been reported to inhibit only extracellular CAs in a variety ofarthropods (such as the crab Callinectes sapidus) and fish [57]. Themain draw-back of QAS is its high toxicity in higher vertebrates [57].

Enzyme activity of carbonic anhydrases (including that of CA IX) can beefficiently blocked by sulfonamide inhibitors. That fact has beentherapeutically exploited in diseases caused by excessive activities ofcertain CA isoforms (e.g. CA II in glaucoma). There is also anexperimental evidence that sulfonamides may block tumor cellproliferation and invasion in vitro and tumor growth in vivo, but thetargets of those sulfonamides have not been identified yet. However, thesulfonamides available so far indiscriminately inhibit various CAisoenzymes (14 are presently known in humans) that are localized indifferent subcellular compartments and play diverse biological roles.This lack of selectivity compromises the clinical utilization of thesecompounds (due to undesired side effects caused by concurrent inhibitionof many CA isoforms) and represents a main drawback also for thesulfonamide application against CA IX in anticancer therapy.

Thus, there is a need in the art for membrane-impermeant, potent CA IXinhibitors, which would become doubly selective inhibitors for CA IX.The inventors have previously made and described some of themembrane-impermeant molecules described here; however, they werecharacterized only for their ability to inhibit CA I, CA II and CA IV.While others have studied effects of selective inhibition ofextracellular CA by membrane impermeant agents in retinal prigmentedepithelia or muscle [34, 120], these agents have not been characterizedfor their ability to inhibit CA IX. Since CA IX is one of the fewextracellular carbonic anhydrases, a membrane-impermeant selectiveinhibitor of CA IX would be doubly selective for this enzyme and therebyavoid side effects associated with nonspecific CA inhibition.

SUMMARY OF THE INVENTION

The inventors have shown that MN/CA IX contributes to acidification ofextracellular pH in hypoxia but not in normoxia. MN/CA IX-selectivesulfonamides are shown to reduce the medium acidification and to bindonly to hypoxic cells containing the wild type MN/CA IX. MN/CA IX'scontributing to the acidification of the hypoxic extracellular milieu isconsidered to have important implications for the development of cancer.The disclosed experimental results indicate that hypoxia up-regulatesboth the expression level and enzyme activity of MN/CA IX, that is,hypoxia activates the CA catalytic activity of MN/CA IX. That is a veryimportant finding because intratumoral hypoxia is a clinically relevantfactor increasing aggressiveness of tumor cells and reducing success oftherapy.

The invention concerns in one aspect diagnostic/prognostic andtherapeutic methods for preneoplastic/neoplastic disease associated withabnormal MN/CA IX expression, comprising the use of MN/CA IX-specificinhibitors which bind preferentially to the activated form of the CAdomain of MN/CA IX, and not to the inactive form of the CA domain ofMN/CA IX. Preferred inhibitors according to the methods of the inventionare activated MN/CA IX-specific inhibitors which are labeled, and whichcan be used to identify regions of hypoxic MN/CA IX expression, and notnon-hypoxic MN/CA IX expression. Exemplary activated MN/CA IX-specificinhibitors include the sulfonamide Compounds 5, 6, 39 and 92, whosestructures are shown in FIGS. 4A and 8A.

Further, MN/CA IX-specific inhibitors which are useful according to themethods of the invention may comprise any molecules that preferentiallybind only the activated form of the CA domain of MN/CA IX, and not theinactive form of the CA domain of MN/CA IX. Such molecules may beorganic or inorganic, preferably organic molecules. Such organicmolecules may be sulfonamides or antibodies which selectively bind theactivated form of the CA domain of MN/CA IX. Preferred organic moleculesinclude monoclonal antibodies which specifically bind the activated formof the CA domain of MN/CA IX.

In one aspect, the invention concerns a diagnostic/prognostic method fora preneoplastic/neoplastic disease associated with abnormal MN/CA IXexpression, comprising determining whether MN/CA IX is activated in avertebrate sample, comprising a) contacting said sample with a specificinhibitor of activated MN/CA IX, and b) detecting or detecting andquantifying binding of said specific inhibitor of activated MN/CA IX insaid sample; wherein binding of said inhibitor to said MN/CA IXindicates that said MN/CA IX is activated, preferably wherein saidactivated MN/CA IX is hypoxia-activated.

Preferably, said specific inhibitor of activated MN/CA IX is an MN/CAIX-specific sulfonamide or an MN/CA IX-specific antibody. Preferablysaid specific sulfonamide inhibitor of activated MN/CA IX is an aromaticsulfonamide or a heterocyclic sulfonamide. Alternatively, saidsulfonamide specific inhibitor of activated MN/CA IX is amembrane-impermeant pyridinium derivative of an aromatic sulfonamide ora membrane-impermeant pyridinium derivative of a heterocyclicsulfonamide. Also preferably, said MN/CA IX-specific sulfonamide isselected from the group consisting of Compounds 1-92, whose structuresare shown in Tables 2 and 3, and/or FIGS. 4 and 8A. Further preferably,said MN/CA IX-specific sulfonamide is selected from the group consistingof Compounds 5, 6, 39, or 92.

Said specific inhibitor of activated MN/CA IX can be conjugated to alabel or a visualizing means, preferably fluorescein isothiocyanate,wherein said detecting or detecting and quantifying binding comprisesdetecting or detecting and quantifying said label or said visualizingmeans on cells in said sample, and wherein said detecting or saiddetecting and quantifying at a level above that for a control sample isindicative of hypoxic precancerous or cancerous cells that abnormallyexpress activated MN/CA IX in said sample. Said method may furthercomprise detecting the binding of an antibody that specifically binds toa domain of the MN/CA IX protein other than the carbonic anhydrasedomain.

Another exemplary method that is diagnostic or diagnostic and prognosticfor precancer and/or cancer comprises contacting a mammalian sample witha MN/CA IX-specific inhibitor conjugated to a label or a visualizingmeans, and detecting or detecting and quantifying binding of said MN/CAIX-specific inhibitor to cells in said sample by detecting or detectingand quantifying said label or said visualizing means on cells in saidsample, wherein said detection or said detection and quantitation at alevel above that for a control sample is indicative of precancerous orcancerous cells that overexpress MN/CA IX in said sample. Such a methodcan be of particular diagnostic and prognostic importance by detectingor detecting and quantitating MN/CA IX activated by hypoxic conditions.Hypoxia combined with MN/CA IX overepression indicates that the mammalfrom whom the sample was taken is considered to have a poorer prognosis,and decisions on treatment for said mammal are made in view of thepresence of said hypoxic conditions.

MN/CA IX as a hypoxia marker is useful in general in making therapeuticdecisions. For example, a cancer patient whose tumor is known to expressMN/CA IX at an abnormally high level would not be a candidate forcertain kinds of chemotherapy and radiotherapy, but would be a candidatefor hypoxia-selective chemotherapy.

In one embodiment of the invention, MN/CA IX-specific inhibitors areused in methods that aid in selecting patient therapy, for example, in amethod wherein the inhibitor's binding to activated MN/CA IX isdetectable at a level above that for a control sample, andhypoxia-selective therapy is selected. Preferably such hypoxia-selectivetherapy comprises the use of drugs that are toxic only under hypoxicconditions, for example, wherein the therapy comprises the use oftirapazamine or AQ4N. In another embodiment of the invention, theinhibitor's binding to activated MN/CA IX is not detectable at a levelabove that for a control sample, and the therapy consequently selectedis radiotherapy and/or non-hypoxia-selective chemotherapy.

In another aspect, the invention concerns a method of imaging hypoxictissues in a patient, comprising a) administering to said patient aspecific inhibitor of activated MN/CA IX, said inhibitor linked to animaging agent; and b) detecting the binding of said inhibitor. Saidspecific inhibitor of activated MN/CA IX is preferably an MN/CAIX-specific sulfonamide or an MN/CA IX-specific antibody. Morepreferably, said MN/CA IX-specific sulfonamide is an aromatic or aheterocyclic sulfonamide, and said MN/CA IX-specific antibody is amonoclonal antibody.

Still another aspect of the invention concerns a method of therapy for apreneoplastic/neoplastic disease associated with hypoxic tissues,comprising administering a specific inhibitor of activated MN/CA IX,preferably an MN/CA IX-specific sulfonamide. Preferably, said specificinhibitor of activated MN/CA IX is an aromatic sulfonamide or aheterocyclic sulfonamide. Alternatively, said specific inhibitor ofactivated MN/CA IX is preferably a membrane-impermeant pyridiniumderivative of an aromatic sulfonamide or a membrane-impermeantpyridinium derivative of a heterocyclic sulfonamide. More preferably,said MN/CA IX-specific sulfonamide is selected from the group consistingof Compounds 1-92. Most preferably, said MN/CA IX-specific sulfonamideis selected from the group consisting of Compounds 5, 6, 39, or 92.

Said specific inhibitor of activated MN/CA IX can also be an MN/CAIX-specific antibody, alone or conjugated to a toxic and/or cytostaticagent; preferably said MN/CA IX-specific antibody is a monoclonalantibody.

In another embodiment of the invention, the method of therapy comprisesthe use of a specific inhibitor of activated MN/CA IX conjugated to avector comprising a gene that expresses a cytotoxic protein. Said vectormay further comprise a MN/CA9 promoter or MN/CA9 promoter fragment,and/or one or more hypoxia response elements.

In still another embodiment of the invention, the method of therapycomprises the use of a specific inhibitor of activated MN/CA IX tomodulate the efficiency of chemotherapeutic drugs whose uptake oractivity is pH-dependent.

The instant invention is related to (1) the recognition that certaincarbonic anhydrase inhibitors (CAIs), preferably sulfonamides,selectively target the cancer-related, hypoxia-induced MN/CA IX; (2) theuse of such CAIs, preferably sulfonamides, as lead compounds for thedesign and synthesis of MN/CA IX-specific inhibitors; (3) the employmentof said MN/CA IX-specific inhibitors for anticancer therapy based uponthe inhibition of MN/CA IX-mediated acidification of tumormicroenvironments; and (4) the use of the specificity of potent MN/CAIX-specific inhibitors for diagnostic/prognostic methods includingimaging methods, such as scintigraphy, and for gene therapy. Theinvention is particularly directed to the use of MN/CA IX-specificinhibitors for the development of drugs possessing anticancer propertiesand to modulate conventional chemotherapy for preneoplastic andneoplastic disease characterized by MN/CA IX expression, particularlyMN/CA IX overexpression.

In one aspect, the invention concerns methods of treating a mammal for apre-cancerous or cancerous disease, wherein said disease ischaracterized by overexpression of MN/CA IX protein, comprisingadministering to said mammal a therapeutically effective amount of acomposition comprising a compound, wherein said compound is selectedfrom the group consisting of organic and inorganic molecules, andwherein said compound is determined to be a potent inhibitor of MN/CA IXenzymatic activity in a screening assay to determine the K_(I) of acompound inhibiting the enzymatic activity of MN/CA IX, wherein if saidinhibition constant K_(I) is determined to be less than about 50nanomolar, said compound is determined be a potent inhibitor of MN/CA IXenzymatic activity; and wherein said compound is not selected from thegroup consisting of acetazolamide, ethoxzolamide, methazolamide andcyanate. Said mammal is preferably human, and said K_(I) is preferablyless than about 35 nanomolar, more preferably less than about 25nanomolar, and still more preferably less than about 10 nanomolar.Exemplary enzymatic screening assays that can be used to determine theK, of a compound inhibiting the enzymatic activity of MN/CA IX aredescribed below under “Enzyme Assays” in the Materials and Methodssection, and also described in references cited in Table 1, which arehereby incorporated by reference.

Such methods can also be framed as methods of treating precancer and/orcancer, or inhibiting the growth of precancerous and/or cancerous cellsin a mammalian subject, wherein said precancer and cancer arecharacterized by the overexpression of MN/CA IX. Said methods can alsobe framed as inhibiting the growth of such precancerous or cancerousmammalian cells overexpressing MN/CA IX comprising contacting said cellswith a MN/CA IX-specific inhibitor of this invention.

The MN/CA IX-specific inhibitors of this invention can be administeredin a therapeutically effective amount, preferably dispersed in aphysiologically acceptable nontoxic liquid vehicle. Different routes ofadministration may be preferred depending on the site or type ofpreneoplastic/neoplastic disease, for example, solid or non-solid tumoror metastasis. In general, parenteral administration would be preferredto avoid undesired effects of systemic treatment, for example, thosethat could be occasioned by binding of the inhibitors to thegastrointestinal mucosa. Injection into or into the vicinity of thepreneoplastic/neoplastic disease would be generally preferred. Forexample, such injections could be intravenous, intraperitoneal, rectal,subcutaneous, intramuscular, intraorbital, intracapsular, intraspinal,intrasternal, intramedullary, intralesional, intradermal, among otherroutes of injection. Also, other modes of administration, for example,by suppository or topically, can be used as would be appropriate to thetarget disease. The pharmaceutical formulation would be designed inaccordance with known standards as suitable for the route ofadministration.

Said MN/CA IX-specific inhibitors are preferably organic, morepreferably aromatic or heterocyclic, and still more preferably anaromatic sulfonamide or a heterocyclic sulfonamide. Said aromaticsulfonamide may be a substituted aromatic sulfonamide, wherein saidaromatic sulfonamide comprises an aromatic ring structure bearing asulfonamide moiety bonded to said ring structure and optionally bearingone or more substituents independently selected from the groupconsisting of halogeno, nitro, and an alkylamino group, wherein thealkyl radical of said alkylamino group comprises 1 to 4 carbon atoms.

Preferably the MN/CA IX-specific inhibitors of this invention are morepotent inhibitors of MN/CA IX enzymatic activity than of the enzymaticactivity of a carbonic anhydrase selected from the group consisting ofCA I, CA II and CA IV. More preferably, the MN/CA IX-specific inhibitorsare more potent inhibitors of MN/CA IX enzymatic activity than of theenzymatic activity of at least two carbonic anhydrases selected from thegroup consisting of CA I, CA II and CA IV. Still more preferably, theMN/CA IX-specific inhibitors are more potent inhibitor of MN/CA IXenzymatic activity than of the enzymatic activity of each of thecarbonic anhydrases in the group consisting of CA I, CA II and CA IV.

However, since CA II is a particularly abundant and significant CA, thatis cytosolic, it is important when the MN/CA IX-specific inhibitors ofthis invention are not membrane-impermeant, that they may be more potentinhibitors of MN/CA IX enzymatic activity than of the enzymatic activityof CA II. Exemplary enzymatic screening assays to determine the K_(I) ofCA II inhibitors are described below under “Enzyme Assays” in theMaterials and Methods section, and also described in references cited inTable 1, which are hereby incorporated by reference.

Exemplary and preferred aromatic sulfonamide or heterocyclic sulfonamideMN/CA IX-specific inhibitors of this invention are selected from thegroup consisting of Compounds 1-26 shown in FIG. 4, and theirFITC-derivatives. Exemplary preferred aromatic sulfonamide MN/CAIX-specific aromatic sulfonamides are Compounds 1, 6, and 23-26. Apreferred aromatic sulfonamide MN/CA IX-specific inhibitor can be thatwherein a halogen atom is bonded to at least one carbon atom in thearomatic ring of said aromatic sulfonamide. Particularly preferredaromatic sulfonamide MN/CA IX-specific inhibitors are selected from thegroup consisting of Compounds 5 and 6, and the FITC-derivative ofCompound 5, Compound 92 (whose structure is shown in FIG. 8A).Particularly preferred heterocyclic sulfonamide MN/CA IX-specificinhibitors are Compounds 14, 15, 21 and 22.

Preferred heterocyclic sulfonamide MN/CA IX-specific inhibitors can besubstituted heterocyclic sulfonamides, wherein said substitutedheterocyclic sulfonamide comprises a heterocyclic ring structure bearinga sulfonamide moiety bonded to said ring structure and optionallybearing one or more substituents independently selected from a groupconsisting of halogeno, nitro, and an alkylamino group, wherein thealkyl radical of said alkylamino group comprises 1 to 4 carbon atoms.Preferred heterocyclic sulfonamide MN/CA IX-specific inhibitors may behalogenated.

Further preferred methods of treating mammals for pre-cancerous orcancerous disease, wherein said disease is characterized byoverexpression of MN/CA IX protein, comprise administering to saidmammal membrane-impermeant MN/CA IX-specific inhibitors. Atherapeutically effective amount of such a membrane-impermeant MN/CAIX-specific inhibitor can be administered in a composition comprisingthe membrane-impermeant compound, wherein said membrane-impermeantinhibitor compound is selected from the group consisting of organic andinorganic molecules, and wherein said membrane-impermeant compound isdetermined to be a potent inhibitor of MN/CA IX enzymatic activity in ascreening assay.

Such a membrane-impermeant MN/CA IX specific inhibitor compound ispreferably organic, and more preferably a pyridinium derivative of anaromatic sulfonamide or a pyridinium derivative of a heterocyclicsulfonamide. Such membrane-impermeant MN/CA IX-specific inhibitorcompounds are preferably more potent inhibitors of MN/CA IX enzymaticactivity than of the enzymatic activity of a carbonic anhydrase selectedfrom the group consisting of CA I, CA II and CA IV, and still morepreferably more potent inhibitors of MN/CA IX enzymatic activity than ofthe enzymatic activity of at least two carbonic anhydrases selected fromthe group consisting of CA I, CA II and CA IV. Further more preferably,said membrane-impermeant MN/CA IX-specific inhibitor compounds are morepotent inhibitors of MN/CA IX enzymatic activity than of the enzymaticactivity of each of the carbonic anhydrases in the group consisting ofCA I, CA II and CA IV. Since both MN/CA IX and CA IV are membrane boundCAs, it is particularly important that the membrane-impermeant MN/CAIX-specific inhibitor compounds are more potent inhibitors of MN/CA IXenzymatic activity than of the enzymatic activity of CA IV.

Exemplary enzymatic screening assays that can be used to determine theK_(I) of a compound inhibiting the enzymatic activity of CA IV aredescribed below under “Enzyme Assays” in the Materials and Methodssection, and also in references cited in Table 1, which are herebyincorporated by reference.

Preferred membrane-impermeant MN/CA IX-specific inhibitor compounds thatare pyridinium derivatives of aromatic sulfonamides are selected fromthe group consisting of sulfanilamide, homosulfanilamide and4-aminoethyl-benzenesulfonamide. Preferred pyridinium derivatives ofaromatic sulfonamides can have the general formula of:

wherein

n is 0, 1, or 2;

R2, R3, R4 and R6 are each independently selected from the groupconsisting of hydrogen, alkyl moieties comprising from 1 to 12 carbonatoms, and aryl moieties. Further preferred pyridinium derivatives ofaromatic sulfonamides are Compounds 27-70 shown in Table 2. Exemplarypreferred pyridinium derivatives of aromatic sulfonamides are Compounds39, 55, 58, 59 and 70. Particularly preferred is Compound 39 shown inTable 2 and FIG. 8A.

When said MN/CA IX-specific inhibitors are membrane-impermeantpyridinium derivatives of a heterocyclic sulfonamides, a preferredcompound is a pyridinium derivative of aminobenzolamide.

Preferred MN/CA IX-specific inhibitor compounds that are pyridiniumderivatives of heterocyclic sulfonamides may have the general formulaof:

wherein R1, R2, R3, R4 and R5 are each independently selected from thegroup consisting of hydrogen, alkyl moieties comprising from 1 to 12carbon atoms, and aryl moieties. Further preferred pyridiniumderivatives of heterocyclic sulfonamides are Compounds 71-91 shown inTable 3.

In another aspect, this invention concerns methods of inhibiting tumorgrowth in a patient having a tumor, the cells of which tumor arecharacterized by overexpression of MN/CA IX protein, comprisingadministering to said patient a therapeutically effective amount of acomposition comprising a compound, wherein said compound is selectedfrom the group consisting of organic and inorganic molecules, andwherein said compound is determined to be a potent inhibitor of MN/CA IXenzymatic activity in a screening assay as outlined above.

In another therapeutic aspect of the invention, the MN/CA IX-specificinhibitors can be conjugated to radioisotopes for administration. Also,the MN/CA IX-specific inhibitors can be administered concurrently and/orsequentially with radiation and/or with a therapeutically effectiveamount in a physiologically acceptable formulation of one or more of thefollowing compounds selected from the group consisting of: conventionalanticancer drugs, chemotherapeutic agents, different inhibitors ofcancer-related pathways, bioreductive drugs, MN/CA IX-specificantibodies and MN/CA IX-specific antibody fragments that arebiologically active. Preferably said MN/CA IX-specific antibodies and/orMN/CA IX-specific antibody fragments are humanized or fully human, andmay be attached to a cytotoxic entity.

In another therapeutic aspect, this invention concerns methods oftreating a mammal for a precancerous or cancerous disease, wherein saiddisease is characterized by overexpression of MN/CA IX protein,comprising administering to said mammal a therapeutically effectiveamount in a physiologically acceptable formulation of a vectorconjugated to a potent MN/CA IX-specific inhibitor, wherein said vectorexpresses a wild-type gene that is absent from or mutated in a MN/CA IXexpressing cell, that is precancerous or cancerous, and wherein the wildtype gene product has an anticancer effect in said cell; or wherein saidvector comprises a gene that expresses a cytotoxic protein. An exemplarywild-type gene would be the von Hippel-Lindau gene known to be directlyinvolved in the constitutive expression of MN/CA IX in renal cellcarcinoma.

Preferably said vector comprises a MN/CA IX promoter or a MN/CA IXpromoter fragment, wherein said promoter or promoter fragment comprisesone or more hypoxia response elements (HREs), and wherein said promoteror promoter fragment is operably linked to said wild-type gene or tosaid gene that expresses a cytotoxic protein. Preferably the MN/CAIX-specific inhibitor conjugated to the vector has a K_(I) as determinedin a CO₂ saturation assay to be less than about 50 nM, more preferablyless than about 35 nM, still more preferably less than about 25 nM andstill further more preferably less than about 10 nM. Preferably, saidpotent MN/CA IX inhibitor is not selected from the group consisting ofacetazolamide, ethoxzolamide, methazolamide and cyanate.

Brown, J. M. [16] points out at page 157 that “solid tumours areconsiderably less well oxygenated than normal tissues. This leads toresistance to radiotherapy and anticancer chemotherapy, as well aspredisposing to increased tumour metastases.” Brown explains how tumorhypoxia can be exploited in cancer treatment. One strategy to exploittumor hypoxia for cancer treatment proposed by Brown [16] is to usedrugs that are toxic only under hypoxic conditions. Exemplary andpreferred drugs that could be used under that strategy includetirapazamine and AQ4N, a di-N-oxide analogue of mitozantrome.

A second mode of exploiting hypoxia proposed by Brown [16] is by genetherapy strategies developed to take advantage of the selectiveinduction of is HIF-1. Brown notes that a tumor-specific delivery systemcan be developed wherein a promoter that is highly responsive to HIF-1would drive the expression of a conditionally lethal gene under hypoxicbut not normoxic conditions. The MN/CA IX promoter is just such apromoter highly responsive to hypoxia, as well as MN/CA IX promoterfragments comprising one or more HREs. “Expression of an enzyme notnormally found in the human body could, under the control of ahypoxia-responsive promoter [the MN/CA IX promoter], convert a nontoxicpro-drug into a toxic drug in the tumour.” [Brown [16], page 160.]Exemplary is the use of the bacterial cytosine deaminase, which convertsthe nontoxic 5-fluorocytosine to the anticancer drug 5-fluorouracil(5FU) cited by Brown to Trinh et al. [109].

Ratcliffe et al., U.S. Pat. Nos. 5,942,434 and 6,265,390 explain howanti-cancer drugs become activated under hypoxia [119], but that the useof a drug activation system, wherein the enzyme that activates the drugis significantly increased under hypoxia, results in much enhancedtherapeutic effect.

This invention further concerns methods for imaging tumors and/ormetastases that express MN/CA IX in a patient comprising theadministration of a MN/CA IX-specific inhibitor linked to an imagingagent to said patient. A preferred imaging method would encompassscintigraphy.

The assays of this invention are both diagnostic and/or prognostic,i.e., diagnostic/prognostic. The term “diagnostic/prognostic” is hereindefined to encompass the following processes either individually orcumulatively depending upon the clinical context: determining thepresence of disease, determining the nature of a disease, distinguishingone disease from another, forecasting as to the probable outcome of adisease state, determining the prospect as to recovery from a disease asindicated by the nature and symptoms of a case, monitoring the diseasestatus of a patient, monitoring a patient for recurrence of disease,and/or determining the preferred therapeutic regimen for a patient. Thediagnostic/prognostic methods of this invention are useful, for example,for screening populations for the presence of neoplastic orpre-neoplastic disease, determining the risk of developing neoplasticdisease, diagnosing the presence of neoplastic and/or pre-neoplasticdisease, monitoring the disease status of patients with neoplasticdisease, and/or determining the prognosis for the course of neoplasticdisease.

The present invention is useful for treating and for screening thepresence of a wide variety of preneoplastic/neoplastic diseasesincluding carcinomas, such as, mammary, colorectal, urinary tract,ovarian, uterine, cervical, endometrial, squamous cell and adenosquamouscarcinomas; head and neck cancers; mesodermal tumors, such as,neuroblastomas and retinoblastomas; sarcomas, such as osteosarcomas andEwing's sarcoma; and melanomas. Of particular interest are gynecologicalcancers including ovarian, uterine, cervical, vaginal, vulval andendometrial cancers, particularly ovarian, uterine cervical andendometrial cancers. Also of particular interest are cancers of thebreast, of gastrointestinal tract, of the stomach including esophagus,of the colon, of the kidney, of the prostate, of the liver, of theurinary tract including bladder, of the lung, and of the head and neck.Gynecologic cancers of particular interest are carcinomas of the uterinecervix, endometrium and ovaries; more particularly such gynecologiccancers include cervical squamous cell carcinomas, adenosquamouscarcinomas, adenocarcinomas as well as gynecologic precancerousconditions, such as metaplastic cervical tissues and condylomas.

The invention provides methods and compositions for evaluating theprobability of the presence of malignant or pre-malignant cells, forexample, in a group of cells freshly removed from a host. Such an assaycan be used to detect tumors, quantitate their growth, and help in thediagnosis and prognosis of disease. The assays can also be used todetect the presence of cancer metastasis, as well as confirm the absenceor removal of all tumor tissue following surgery, cancer chemotherapyand/or radiation therapy. It can further be used to monitor cancerchemotherapy and tumor reappearance.

The presence of MN antigen can be detected and/or quantitated using anumber of well-defined diagnostic assays. Those in the art can adapt anyof the conventional immunoassay formats to detect and/or quantitate MNantigen as herein disclosed. The immunoassays of this invention can beembodied in test kits which comprise the potent MN/CA IX-specificinhibitors of this invention, appropriately labeled and/or linked to avisualizing means, as known in the art. Such test kits can be in solidphase formats, but are not limited thereto, and can also be in liquidphase format, and can be based on immunohistochemical assays, ELISAS,particle assays, radiometric or fluorometric assays either unamplifiedor amplified, using, for example, avidin/biotin technology, among otherassay formats.

Exemplary MN/CA IX-specific inhibitors of the invention are shown hereinto treat transfected cells that constitutively express MN/CA IX comparedto non-transfected cells with no MN/CA IX expression. The exemplaryMN/CA IX-specific inhibitors are shown to inhibit acidification ofextracellular pH induced by MN/CA IX in cell cultures exposed tohypoxia.

Further, labeled exemplary MN/CA IX-specific inhibitors, such as labeledsulfonamides, for example, conjugated to fluorescein isothiocyanate(FITC), are shown to bind to the surface of MN/CA IX transfected cells,and not to control cells, only in hypoxia but not in normoxia. Thoseexperiments confirm that MN/CA IX-specific inhibitors, such as thesulfonamide compounds described herein, can specifically target MN/CA IXunder conditions characteristic of intratumoral microenvironments.

The MN/CA IX-specific inhibitors of this invention can be useddiagnostically and prognostically for precancer and cancer, and todetermine the status of a patient, and therapeutically, individually orin different combinations with conventional therapeutic regimens totreat precancers and/or cancer. The MN/CA IX-specific inhibitors mayalso be used in cancer research.

More particularly for treating precancer and/or cancer, the MN/CAIX-specific inhibitors of this invention can be used to hinder cancerexpansion and/or progression by blocking MN/CA IX activity. The MN/CAIX-specific inhibitors can be conjugated to radioisotopes forradiotherapy. The MN/CA IX-specific inhibitors can be combined withMN/CA IX-specific antibodies and a variety of conventional therapeuticdrugs, different inhibitors of cancer-related pathways, bioreductivedrugs, and/or radiotherapy, wherein different combinations of treatmentregimens with the MN/CA IX-specific inhibitors of this invention mayincrease overall treatment efficacy. Particularly, the MN/CA IX-specificinhibitors of this invention may be combined with therapy using MN/CAIX-specific antibodies and/or MN/CA IX-specific antibody fragments,preferably humanized MN/CA IX-specific antibodies and/or biologicallyactive fragments thereof, and more preferably fully human MN/CAIX-specific antibodies and/or fully human MN/CA IX-specific biologicallyactive antibody fragments. Said MN/CA IX-specific antibodies andbiologically active MN/CA IX-specific antibody fragments, preferablyhumanized and more preferably fully human, may be conjugated to acytotoxic entity, for example, a cytotoxic protein, such as ricin A,among many other cytotoxic entities.

Still further, a MN/CA IX-specific inhibitor of this invention could becoupled to a vector for targeted delivery to MN/CA IX-specificexpressing cells for gene therapy (for example, with the wild-type vonHippel-Lindau gene), or for effecting the expression of cytotoxicproteins, preferably wherein said vector comprises a MN/CA IX promoteror MN/CA IX promoter fragment comprising the MN/CA IX hypoxia responseelement (HRE) or a HRE of another gene, and more preferably wherein theMN/CA IX promoter or MN/CA IX promotor fragment comprises more than oneHRE, wherein said HRE or HREs is or are either of MN/CA IX, and/or ofother genes and/or of genetically engineered HRE consensus sequences ina preferred context.

Particularly, the MN/CA IX-specific inhibitors of this invention can beused diagnostically/prognostically to detect precancerous and/orcancerous cells by binding to MN/CA IX, preferably to MN/CA IX activatedby hypoxic conditions, wherein said MN/CA IX specific inhibitors arecoupled to a label or to some visualizing means. Such detection,particularly of hypoxic conditions, and MN/CA IX overexpression, can behelpful in determining effective treatment options, and in predictingtreatment outcome and the prognosis of disease development. Further theMN/CA IX-specific inhibitors when labeled or linked to an appropriatevisualizing means can be used for imaging tumors and/or metastases thatexpress MN/CA IX.

The MN/CA IX-specific inhibitors of this invention can also be used inbasic and pre-clinical research. For example, the MN/CA IX-specificinhibitors can be used to study the regulation of MN/CA IX enzymeactivity, to study the role of MN/CA IX in tumor growth and metabolism,and to study the role of MN/CA IX in response to treatment by drugs,radiation, inhibitors and other therapeutic regimens.

Further provided are screening assays for compounds that are useful forinhibiting the growth of a vertebrate, preferably mammalian, morepreferably human, preneoplastic or neoplastic cell that abnormallyexpresses MN protein. Said screening assays comprise tests for theinhibition of the enzymatic activity of MN by said compounds. Additionalassays provided herein test said compounds for their cell membraneimpermeance.

Aspects of the instant invention disclosed herein are described in moredetail below.

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ABBREVIATIONS

The following abbreviations are used herein:

-   aa—amino acid-   AAZ—acetazolamide-   AE—anion exchanger-   ATCC—American Type Culture Collection-   ΔCA—deletion mutant of CA IX lacking the catalytic domain-   ΔPG—deletion mutant of CA IX lacking the proteoglycan-like domain-   bp—base pairs-   BRL—Bethesda Research Laboratories-   BRZ—brinzolamide-   BSA—bovine serum albumin-   CA—carbonic anhydrase-   CAI—carbonic anhydrase inhibitor-   CAM—cell adhesion molecule-   CARP—carbonic anhydrase related protein-   Ci—curie-   cm—centimeter-   CNS—central nervous system-   cpm—counts per minute-   C-terminus—carboxyl-terminus-   ° C.—degrees centigrade-   DCP—dichlorophenamide-   DEAE—diethylaminoethyl-   DMEM—Dulbecco modified Eagle medium-   ds—double-stranded-   DZA—dorzolamide-   EDTA—ethylenediaminetetraacetate-   EZA—ethoxzolamide-   F—fibroblasts-   FCS—fetal calf serum-   FITC—fluorescein isothiocyanate-   H—HeLa cells; hypoxia-   HIF—hypoxia inducible factor-   IC—intracellular-   kb—kilobase-   kbp—kilobase pairs-   kd or kDa—kilodaltons-   K_(I)—inhibition constant-   KS—keratan sulphate-   LTR—long terminal repeat-   M—molar-   mA—milliampere-   MAb—monoclonal antibody-   ME—mercaptoethanol-   MEM—minimal essential medium-   min.—minute(s)-   mg—milligram-   ml—milliliter-   mM—millimolar-   MMC—mitomycin C-   mmol—millimole-   MZA—methazolamide-   N—normal concentration; normoxia-   NEG—negative-   ng—nanogram-   nm—nanometer-   nM—nanomolar-   nt—nucleotide-   N-terminus—amino-terminus-   ODN—oligodeoxynucleotide-   ORF—open reading frame-   PA—Protein A-   PBS—phosphate buffered saline-   PCR—polymerase chain reaction-   PG—proteoglycan-   pHe—extracellular pH-   pI—isoelectric point-   PMA—phorbol 12-myristate 13-acetate-   POS—positive-   PVDF—polyvinylidene difluoride-   pVHL—von Hippel-Lindau tumor suppressor protein-   Py—pyrimidine-   QAS—quaternary ammonian sulfonilamide-   QSAR—quantitative structure-activity relationship(s)-   RACE—rapid amplification of cDNA ends-   RCC—renal cell carcinoma-   RIA—radioimmunoassay-   RIP—radioimmunoprecipitation-   RIPA—radioimmunoprecipitation assay-   RNP—RNase protection assay-   RT-PCT—reverse transcription polymerase chain reaction-   SAC—Staphylococcus aureus cells-   SAR—structure-activity relationship-   sc—subcutaneous-   SDS—sodium dodecyl sulfate-   SDS-PAGE—sodium dodecyl sulfate-polyacrylamide gel electrophoresis-   SINE—short interspersed repeated sequence-   SP—signal peptide-   SP-RIA—solid-phase radioimmunoassay-   TBE—Tris-borate/EDTA electrophoresis buffer-   TC—tissue culture-   TCA—trichloroacetic acid-   TC media—tissue culture media-   tk—thymidine kinase-   TM—transmembrane-   Tris—tris (hydroxymethyl) aminomethane-   μCi—microcurie-   μg—microgram-   μl—microliter-   μM—micromolar

Cell Lines

-   BL21 (DE3)—Escherichia coli strain described by Lindskog's group    (for CA I, II expression) [Lindskog et al., “Structure-function    relations in human carbonic anhydrase II as studied by site-directed    mutagenesis,” in Carbonic anhydrase—From biochemistry and genetics    to Physiology and clinical medicine, Botre et al., Eds., VCH,    Weinheim, pp. 1-13 (1991)]-   BL21-GOLD—Escherichia coli strain (from Stratagene) used for CA IX    (DE3) expression)-   HeLa—cell line derived from human cervical adenocarcinoma; cells    normally express endogenous CA IX-   HeLa—ΔCA—HeLa cells stably transfected with recombinant plasmids to    contain ΔCA but not CA IX under normoxia, and express both proteins    under hypoxia, which apparently form mixed oligomers (composed of    both CA IX and ΔCA)-   HeLa-mock—HeLa cells cotransfected with empty pSG5C and pSV2 neo    plasmids as negative controls-   MDCK—cell line derived from normal canine tubular kidney epithelium;    cells do not express endogenous CA IX-   MDCK-CA IX—MDCK cells stably transfected with recombinant plasmids    to express CA IX constitutively-   MDCK-mock—MDCK cells cotransfected with empty pSG5C and pSV2 neo    plasmids as negative controls-   SiHa—cell line derived from human cervical squamous cell carcinoma;    cells normally express endogenous CA IX

Nucleotide and Amino Acid Sequence Symbols

The following symbols are used to represent nucleotides herein:

Base Symbol Meaning A adenine C cytosine G guanine T thymine U uracil Iinosine M A or C R A or G W A or T/U S C or G Y C or T/U K G or T/U V Aor C or G H A or C or T/U D A or G or T/U B C or G or T/U N/X A or C orG or T/U

There are twenty main amino acids, each of which is specified by adifferent arrangement of three adjacent nucleotides (triplet code orcodon), and which are linked together in a specific order to form acharacteristic protein. A three-letter or one-letter convention is usedherein to identify said amino acids, as, for example, in FIG. 1 asfollows:

3 Ltr. 1 Ltr. Amino acid name Abbrev. Abbrev. Alanine Ala A Arginine ArgR Asparagine Asn N Aspartic Acid Asp D Cysteine Cys C Glutamic Acid GluE Glutamine Gln Q Glycine Gly G Histidine His H Isoleucine Ile I LeucineLeu L Lysine Lys K Methionine Met M Phenylalanine Phe F Proline Pro PSerine Ser S Threonine Thr T Tryptophan Trp W Tyrosine Tyr Y Valine ValV Unknown or other X

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A-C provides the nucleotide sequence for MN/CA IX full-length cDNA[SEQ ID NO: 1]. FIG. 1 A-C also sets forth the predicted amino acidsequence [SEQ ID NO: 2] encoded by the cDNA.

FIG. 2A-F provides a 10,898 bp complete genomic sequence of MN/CA9 [SEQID NO: 3]. The base count is as follows: 2654 A; 2739 C; 2645 G; and2859 T. The II exons are in general shown in capital letters, but exon 1is considered to begin at position 3507 as determined by RNaseprotection assay.

FIG. 3 provides an exon-intron map of the human MN/CA9 gene. Thepositions and sizes of the exons (numbered, cross-hatched boxes), Alurepeat elements (open boxes) and an LTR-related sequence (firstunnumbered stippled box) are adjusted to the indicated scale. The exonscorresponding to individual MN/CA IX protein domains are enclosed indashed frames designated PG (proteoglycan-like domain), CA (carbonicanhydrase domain), TM (transmembrane anchor) and IC (intracytoplasmictail). Below the map, the alignment of amino acid sequences illustratesthe extent of homology between the MN/CA IX protein PG region (aa53-111) [SEQ ID NO: 4] and the human aggrecan (aa 781-839) [SEQ ID NO:5].

FIG. 4 A-B shows the chemical structures of the 26 different sulfonamidecompounds tested in Example 1.

FIG. 5 shows the scheme for the general synthesis of compounds 71-91 ofExample 3 (Scheme 1).

FIG. 6 shows the scheme for the reaction between a pyrylium salt and anamine (Scheme 2), as described in Example 3.

FIG. 7 (discussed in Example 4) illustrates the CA IX-mediatedacidification of the extracellular pH in hypoxia. Values of pHe [FIG.7A] and lactate [FIG. 7B] concentrations are shown in histograms (meanvalues and standard deviations) for cells grown in the constant mediumvolumes, maintained in normoxia (N, 21% O₂) or exposed to hypoxia (H, 2%O₂) for 48 hours. The cells tested were CA IX-transfected MDCK cells andmock-transfected controls for comparison. Five independent experimentswith three different clones of the transfectants and three paralleldishes for each clone were performed.

FIG. 8 (discussed in Example 5) shows sulfonamide inhibition and bindingto hypoxic MDCK-CA IX cells. FIG. 8A shows the chemical structures ofthe CA IX-selective inhibitors used in Examples 5-8: Compound 6[4-(2-aminoethyl)-benzenesulfonamide], Compound 39[4-(2,4,6-trimethylpyridinium-N-methylcarboxamido)-benzensulfonamideperchlorate], and Compound 92 [FITC derivative of homosulfanilamide(Compound 5)]. [FIG. 8B] The sulfonamides were added to MDCK-CA IX cellsjust before the cells were transferred to hypoxia, and pHe was measured48 hours later. At least three independent experiments with threeparallel dishes per sample were performed for each inhibitor. Data areexpressed as differences between the pH values (ΔpH) measured in theuntreated versus treated cells and include the standard deviations.

FIG. 9 (discussed in Example 6) shows the expression and acidificationcapability of the CA IX deletion mutants. FIG. 9A is a schematic drawingof the domain composition of the wild-type (wt) CA IX with the aminoacid positions indicating an extent of deletions in the N-terminal PGdomain (ΔPG) and the central CA domain (ΔCA): SP, signal peptide; PG,proteoglycan-like region; CA, carbonic anhydrase domain; TM,transmembrane anchor; IC, intracytoplasmic tail. [FIG. 9B] ExtracellularpH and production of lactate in the transfected MDCK cells. At leastthree independent experiments were performed using three clonal celllines for each transfected variant with at least three parallel dishes.Data are expressed as mean differences in the pH values (ΔpH) and in thelactate concentrations (Δmg/ml), respectively.

FIG. 10 (discussed in Example 7) shows treatment of the tumor cells byCompound 92 sulfonamide [FITC derivative of homosulfanilamide (Compound5)]. HeLa and SiHa cervical carcinoma cells were incubated for 48 hoursin normoxia and hypoxia, respectively, either in the absence or in thepresence of 1 mM of the Compound 92 sulfonamide. Mean differences in thepH values determined in the treated versus control dishes are shown onthe histogram with indicated standard deviations. The experiment wasrepeated three times using at least three parallel dishes for eachsample.

FIG. 11 (discussed in Example 8) illustrates ectopic expression of ΔCAmutant in HeLa cells. Values of pHe in the culture media of HeLa cellstransfected with ΔCA in comparison to the mock-transfected controls.Data represent mean differences in the pH values and correspondingstandard deviations. The experiment was repeated three times with threedifferent clones of the transfected HeLa, each having at least threeparallel dishes.

DETAILED DESCRIPTION

The novel methods of the present invention comprise inhibiting thegrowth of tumor cells which overexpress MN protein with compounds thatinhibit the enzymatic activity of MN protein. Said compounds are organicor inorganic, preferably organic, more preferably sulfonamides. Stillmore preferably, said compounds are pyridinium derivatives of aromaticor heterocyclic sulfonamides. These preferred pyridinium derivatives ofsulfonamides are likely to have fewer side effects than other compoundsin three respects: they are small molecules, they aremembrane-impermeant, and they are specific potent inhibitors of theenzymatic activity of the tumor-associated MN/CA IX protein.

The use of oncoproteins as targets for developing new cancertherapeutics is considered conventional by those of skill in the art.[See, e.g., Mendelsohn and Lippman [61]. However, the application ofsuch approaches to MN is new. In comparison to other tumor-relatedmolecules (e.g. growth factors and their receptors), MN has the uniqueproperty of being differentially expressed in preneoplastic/neoplasticand normal tissues, which are separated by an anatomic barrier.

The pyridinium derivatives of sulfonamides of the present invention canbe formed, for example, by creating bonds between pyrylium salts andaromatic or heterocyclic sulfonamide reagents, as described below. Thearomatic or heterocyclic sulfonamide portion of a pyridinium salt of asulfonamide compound can be called the “head,” and the pyridiniumportion can be called the “tail.”

It can be appreciated by those of skill in the art that various othertypes of linkages can couple the pyridinium portion with the sulfonamideportion. It can further be appreciated that alternate methods, inaddition to those disclosed herein, can be used to make the pyridiniumderivatives of the present invention.

As used herein, “cancerous” and “neoplastic” have equivalent meanings,and “precancerous” and “preneoplastic” have equivalent meanings.

As used herein, the term “aromatic” when applied to sulphonamidestructures means “comprising an aromatic ring, without an additionalheterocyclic ring.” The term “heterocyclic” when applied to sulphonamidestructures means “comprising a heterocyclic ring, with or without anadditional aromatic ring.”

As used herein, the term “alkyl”, alone or in combination, refers to astraight-chain or branched-chain alkyl radical containing from 1 to 12,preferably from 1 to 6 and more preferably from 1 to 4, carbon atoms.Examples of such radicals include, but are not limited to, methyl,ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl,pentyl, iso-amyl, hexyl, decyl and the like.

The term “aryl”, alone or in combination, means a phenyl or naphthylradical which optionally carries one or more substituents selected fromalkyl, alkoxy, halogen, hydroxy, amino, nitro, cyano, haloalkyl,carboxy, alkoxycarbonyl, cycloalkyl, heterocycloalkyl, amido, mono anddialkyl substituted amino, mono and dialkyl substituted amido and thelike, such as phenyl, p-tolyl, 4-methoxyphenyl, 4-(tert-butoxy)phenyl,3-methyl-4-methoxyphenyl, 4-fluorophenyl, 4-chlorophenyl, 3-nitrophenyl,3-aminophenyl, 3-acetamidophenyl, 4-acetamidophenyl,2-methyl-3-acetamidophenyl, 2-methyl-3-aminophenyl,3-methyl-4-aminophenyl, 2-amino-3-methylphenyl,2,4-dimethyl-3-aminophenyl, 4-hydroxyphenyl, 3-methyl-4-hydroxyphenyl,1-naphthyl, 2-naphthyl, 3-amino-1-naphthyl, 2-methyl-3-amino-1-naphthyl,6-amino-2-naphthyl, 4,6-dimethoxy-2-naphthyl and the like.

Preferred sulfonamides of the present invention are aromatic andheterocyclic sulfonamides. The structures of representative sulfonamidesof this group, designated 1-26, are shown in FIG. 4.

More preferred sulfonamides of the present invention are pyridiniumderivatives of aromatic sulfonamides and have the general formula (A)below,

wherein n is 0, 1, or 2; and R2, R3, R4 and R6 are each independentlyselected from the group consisting of hydrogen, alkyls and aryls. Thestructures of representative sulfonamides of this group, designated 27through 70, are shown as derivatives of the general structure (A), inTable 2.

Alternatively, more preferred sulfonamides of the present invention arepyridinium derivatives of heterocyclic sulfonamides and have the generalformula (B) below, wherein said pyridinium derivative of a heterocyclicsulfonamide has the general formula of:

wherein R1, R2, R3, R4 and R5 are each independently selected from thegroup consisting of hydrogen, alkyls and aryls. The structures ofrepresentative sulfonamides of this group, designated 71 through 91, areshown as derivatives of the general structure (B), in Table 3.

Representative sulfonamide derivatives of the group of compoundsrepresented by the general formulas (A) and (B) have CA IX inhibitoryactivity, and are potentially useful therapeutically as anticanceragents in treating MN-associated tumors.

Further, biologic activity of the identified sulfonamides will be testedin vitro by inhibition of the carbonic anhydrase enzymatic activity ofthe MN protein, by effects on cell morphology and growth characteristicsof MN-related tumor cells (HeLa) and of control cells [104]. In vivoscreening will be carried out in nude mice that have been injected withHeLa cells.

It can be appreciated by those of skill in the art that various other CAIX-specific inhibitors can be useful according to the methods of theinvention, and may comprise any molecules that preferentially bind onlythe activated form of the CA domain of CA IX, and not the inactive formof the CA domain of CA IX. Such molecules may be organic or inorganic,preferably organic molecules. Such organic molecules may be antibodies,preferably monoclonal antibodies, which selectively bind the activatedform of the CA domain of CA IX. For example, monoclonal antibodies havebeen described which specifically recognize the epitope of caspases thatis characteristic of the activated form of those proteases [143].Therefore, activation of the CA domain of CA IX could theoretically bedetected both indirectly from tumor cell samples and directly in situ,immunocytochemically.

Representative Sulfonamide Inhibitors of CA IX

The sulfonamides investigated in Example 1 for the inhibition of thetumor-associated isozyme CA IX, of types 1-26 are shown in FIG. 4A-B.Compounds 1-6, 11-12, 20 and 26 are commercially available, whereas 7-10[43], 13-19 [24, 90, 97] and 21-25 [79] were prepared as reportedearlier. The six clinically used compounds were also assayed. ForExample 2 compounds (pyridinium derivatives of aromatic sulfonamides),reaction of sulfanilamide, homosulfanilamide or4-(2-aminoethyl)-benzenesulfonamide with 2,6-di-, 2,4,6-tri- or2,3,4,6-tetrasubstituted pyrylium salts afforded the pyridinium salts27-70 investigated here, by the general Bayer—Piccard synthesis [9, 10,97].

As described in Example 3, a series of positively-charged sulfonamides,designated here as compounds 71-91, were obtained by reaction ofaminobenzolamide(5-(4-aminobenzenesulfonylamino)-1,3,4-thiadiazole-2-sulfonamide) withtri-/tetra-substituted pyrilium salts possessing alkyl-, aryl- orcombinations of alkyl and aryl groups at the pyridinium ring (describedbelow). Three of these compounds (71, 75, and 87) have been describedelsewhere [25, 85]; all other compounds of this series are new.

Heterocyclic Sulfonamide Inhibitors of CA IX: Synthesis of PyridiniumDerivatives of Aminobenzolamide

Chemistry: Reaction of aminobenzolamide(5-(4-aminobenzenesulfonylamino)-1,3,4-thiadiazole-2-sulfonamide) [97]with 2,6-di-, 2,4,6-tri- or 2,3,4,6-tetrasubstituted pyrylium saltsafforded the pyridinium salts 71-91 investigated here, by the generalsynthesis of such derivatives with nucleophiles (Scheme 1 as shown inFIG. 5) [6, 26, 108].

Preparation of Compounds: a Large Number of Positively-Chargedsulfonamides, prepared by reaction of amino-sulfonamides with pyryliumsalts [23, 88, 89] were recently reported by this group, and generallytested as inhibitors of the “classical” isozymes CA I, II and IV [81,96, 97, 98]. Based on QSAR studies on several series of CA inhibitors,including some positively-charged derivatives [23, 88, 89], it emergedthat the enhancement of CA inhibitory activity is correlated withincreased positive charges on the heterocyclic/aromatic ringincorporated in such molecules, as well as with “long” inhibitormolecules per se (i.e., molecules extending on the direction passingthrough the Zn(II) ion of the enzyme, the sulfonamide nitrogen atom andthe long axis of the inhibitor) [23, 88, 89]. It appeared thus ofinterest to try to explore this result, designing positively-charged,long sulfonamide CAIs. Thus, we thought of attachingsubstituted-pyridinium moieties to an already potent and long-moleculeCAI suitable for reaction with pyrylium salts, i.e., aminobenzolamide[97]. Indeed, this compound acts as a very potent CAI against isozymesI, II and IV (with inhibition constants in the low nanomolar range—seelater in the text). The substitution pattern of the pyridinium ring waspreviously shown [81, 96, 97, 98] to be critical for the biologicalactivity of this type of sulfonamide CAIs. Thus, a large series of2,4,6-trialkylpyridinium-; 2,6-dialkyl-4-phenylpyridinium-;2-alkyl-4,6-diphenylpyridinium-; 2,4,6-triphenylpyridinium-, togetherwith various 2,6-disubstituted-pyridinium and2,3,5,6-tetrasubstituted-pyridinium aminobenzolamide derivatives havebeen prepared by the reaction described in Scheme 1 (Shown in FIG. 5).

Although apparently simple, the reaction between a pyrylium salt and anamine, leading to pyridinium salts, is in reality a complicated process(Scheme 2, shown in FIG. 6), as established by detailed spectroscopicand kinetic data from Balaban's and Katritzky's groups [6, 26, 108].Thus, the nucleophilic attack of a primary amine RNH₂ on pyryliumcations generally occurs in the α position, with the formation ofintermediates of type IV (depicted in FIG. 6), which by deprotonation inthe presence of bases lead to the 2-amino-tetradehydropyran derivativesV. In many cases the deprotonation reaction is promoted by the amineitself, when this is basic enough (this being the reason why in manycases one works at molar ratios pyrylium:amine of 1:2 when pyridiniumsalts are prepared by this method), or by external catalysts added tothe reaction mixture, such as triethylamine [6, 26, 108]. Thederivatives V are generally unstable, being tautomers with theketodieneamines VI which are the key intermediates for the conversion ofpyryliums into pyridiniums [6, 26, 108]. In acidic media, in therate-determining step of the whole process, ketodieneamines VI may beconverted to the corresponding pyridinium salts VII, although otherproducts, such as vinylogous amides with diverse structures have alsobeen isolated in such reactions [6, 26, 108]. A supplementarycomplication appears when the moiety substituting the 2- and/or6-position(s) of the pyrylium ring is methyl, cases in which aconcurrent cyclisation with formation of the anilines VII in addition tothe pyridinium salts VII, may take place too [6, 26, 108]. Theseconcurrent reactions mentioned above are generally important when theamine to be converted into the pyridinium salt possesses weaknucleophilicity or basicity. This happens to be the case ofaminobenzolamide. In fact, reaction of aminobenzolamide with severalpyrylium salts, performed in a variety of conditions (differentsolvents, such as low molecular weight alcohols (MeOH, EtOH, i-PrOH);DMF; methylene chloride; acetonitrile; diverse molar ratios of thereagents; temperatures from 25 to 150° C.; reaction times between 15 minand 48 hours, etc) led only to the isolation of the unreacted rawmaterials. The only conditions which led to the formation of thepyridinium salts III (depicted in FIG. 5) were the following: anhydrousmethanol in the presence of acetic anhydride as solvent andtriethylamine as catalysts for the deprotonation of the intermediatesIV. Acetic anhydride had the role of reacting with the water formed inthe condensation reaction. This water may in fact act as a competitivenucleophile with aminobenzolamide when reacting with the pyryliumcation, and as a consequence the yields in pyridinium salts woulddramatically be decreased. After the rapid formation of theketodieneamine, catalyzed by triethylamine (and in the presence of theacetic anhydride as water scavenging agent), the cyclisation to thepyridinium ring (the rate-determining step) has been achieved byrefluxation in the presence of acetic acid (2-5 hours). Still the yieldswere not always good, especially for the 2-methyl-containingderivatives.

Representative Sulfonamide Inhibitors of Activated CA IX

In Examples 5 and 7 below, three exemplary CA IX-selective inhibitorstested for extracellular pH effects [Compounds 6, 39 and 92 (theFITC-derivative of Compound 5) as shown in FIG. 8A] bind to CA IXpreferentially under conditions of hypoxia. As indicated in Example 5,all three sulfonamides were able to reduce the extracellularacidification of MDCK-CA IX cells in hypoxia, and their effect on thenormoxic pHe was negligible [FIG. 8]. The FITC-labeled Compound 92 wasdetected only in hypoxic MDCK-CA IX cells, but was absent from theirnormoxic counterparts and from the mock-transfected controls.

Exclusive binding of the FITC-conjugated Compound 92 sulfonamide to thehypoxic cells that express activated CA IX (described in Example 7)offers an attractive possibility for the use of similarsulfonamide-based compounds for imaging purposes in vivo. Moreover, CAIX-selective sulfonamide derivatives may potentially serve as componentsof the therapeutic strategies designed to increase pHe in the tumormicroenvironment and thereby reduce the tumor aggressiveness and thedrug uptake [18, 86, 106, 126].

It is generally accepted that the reaction between CA and inhibitoroccurs principally via a coordination of the ionized inhibitor to thezinc ion through the network of hydrogen bonds with amino acid residuesof the active site, which effectively means that the inhibitor canefficiently bind only to active CA isoforms [102]. It cannot be excludedthat hypoxia influences the conformation and hence the accessibility ofthe active site of CA IX, but this assumption warrants further studies.

Preparation of MN Proteins and/or Polypeptides

The terms “MN/CA IX” and “MN/CA9” are herein considered to be synonymsfor MN. Also, the G250 antigen is considered to refer to MNprotein/polypeptide [112].

Zavada et al., WO 93/18152 and/or WO 95/34650 disclose the MN cDNAsequence shown herein in FIG. 1A-1C [SEQ ID NO: 1], the MN amino acidsequence [SEQ ID NO: 2] also shown in FIG. 1A-1C, and the MN genomicsequence [SEQ ID NO: 3] shown herein in FIG. 2A-2F. The MN gene isorganized into 11 exons and 10 introns.

The first thirty seven amino acids of the MN protein shown in FIG. 1A-1Cis the putative MN signal peptide [SEQ ID NO: 6]. The MN protein has anextracellular domain [amino acids (aa) 38-414 of FIG. 1A-1C [SEQ ID NO:7], a transmembrane domain [aa 415-434; SEQ ID NO: 8] and anintracellular domain [aa 435-459; SEQ ID NO: 9]. The extracellulardomain contains the proteoglycan-like domain [aa 53-111: SEQ ID NO: 4]and the carbonic anhydrase (CA) domain [aa 135-391; SEQ ID NO: 5].

The phrase “MN proteins and/or polypeptides” (MN proteins/polypeptides)is herein defined to mean proteins and/or polypeptides encoded by an MNgene or fragments thereof. An exemplary and preferred MN proteinaccording to this invention has the deduced amino acid sequence shown inFIG. 1. Preferred MN proteins/polypeptides are those proteins and/orpolypeptides that have substantial homology with the MN protein shown inFIG. 1. For example, such substantially homologous MNproteins/polypeptides are those that are reactive with the MN-specificantibodies, preferably the Mab M75 or its equivalent. The VU-M75hybridoma that secretes the M75 Mab was deposited at the ATCC under HB11128 on Sep. 17, 1992.

A “polypeptide” or “peptide” is a chain of amino acids covalently boundby peptide linkages and is herein considered to be composed of 50 orless amino acids. A “protein” is herein defined to be a polypeptidecomposed of more than 50 amino acids. The term polypeptide encompassesthe terms peptide and oligopeptide.

It can be appreciated that a protein or polypeptide produced by aneoplastic cell in vivo could be altered in sequence from that producedby a tumor cell in cell culture or by a transformed cell. Thus, MNproteins and/or polypeptides which have varying amino acid sequencesincluding without limitation, amino acid substitutions, extensions,deletions, truncations and combinations thereof, fall within the scopeof this invention. It can also be appreciated that a protein extantwithin body fluids is subject to degradative processes, such as,proteolytic processes; thus, MN proteins that are significantlytruncated and MN polypeptides may be found in body fluids, such as,sera. The phrase “MN antigen” is used herein to encompass MN proteinsand/or polypeptides.

It will further be appreciated that the amino acid sequence of MNproteins and polypeptides can be modified by genetic techniques. One ormore amino acids can be deleted or substituted. Such amino acid changesmay not cause any measurable change in the biological activity of theprotein or polypeptide and result in proteins or polypeptides which arewithin the scope of this invention, as well as, MN muteins.

The MN proteins and polypeptides of this invention can be prepared in avariety of ways according to this invention, for example, recombinantly,synthetically or otherwise biologically, that is, by cleaving longerproteins and polypeptides enzymatically and/or chemically. A preferredmethod to prepare MN proteins is by a recombinant means. Particularlypreferred methods of recombinantly producing MN proteins are describedbelow. A representative method to prepare the MN proteins shown in FIG.1 or fragments thereof would be to insert the full-length or anappropriate fragment of MN cDNA into an appropriate expression vector asexemplified in the Materials and Methods section.

MN Gene

FIG. 1A-C provides the nucleotide sequence for a full-length MN cDNAclone [SEQ ID NO: 1] isolated as described in Zavada et al., WO95/34650. FIG. 2A-F provides a complete MN genomic sequence [SEQ ID NO:3].

The ORF of the MN cDNA shown in FIG. 1 has the coding capacity for a 459amino acid protein with a calculated molecular weight of 49.7 kd. Theoverall amino acid composition of the MN/CA IX protein is rather acidic,and predicted to have a pI of 4.3. Analysis of native MN/CA IX proteinfrom CGL3 cells by two-dimensional electrophoresis followed byimmunoblotting has shown that in agreement with computer prediction, theMN/CA IX is an acidic protein existing in several isoelectric forms withpIs ranging from 4.7 to 6.3.

The CA domain is essential for induction of anchorage independence,whereas the TM anchor and IC tail are dispensable for that biologicaleffect. The MN protein is also capable of causing plasma membraneruffling in the transfected cells and appears to participate in theirattachment to the solid support. The data evince the involvement of MNin the regulation of cell proliferation, adhesion and intercellularcommunication.

Enzymatic Screening Assays

Assays are provided herein for the screening of compounds for inhibitionof the enzymatic activity of the MN protein. Such assays comprise theincubation of said compound with said MN protein and with a substrateselected from the group consisting of saturated CO₂ and4-nitrophenylacetate, preferably saturated CO₂, and determination of theinhibition constant K_(I) of said compound, wherein said enzymaticactivity of the MN protein is measured by the pH change of an indicatorby stopped flow spectrophotometer.

Screening of representative heterocyclic and aromatic sulfonamides forinhibition of MN protein: From Example 1, it was found that theinhibition profile of isozyme CA IX is very different from that of theclassical isozymes CA I and II (cytosolic) as well as CA IV(membrane-bound). The following particular features may be noted: (i)all the 32 sulfonamides investigated in Example 1 act as CA IXinhibitors, with inhibition constants in the range of 14-285 nM (thecorresponding affinities for the other three isozymes vary in a muchwider range, as seen from data of Table 1). Based on these data, it canbe noted that CA IX is a sulfonamide avid CA, similarly to CA II, theisozyme considered up to now to be responsible for the majority ofpharmacological effect of sulfonamides [22, 29, 83, 93, 94, 95, 102].Still, many other differences are observed between CA IX and otherisozymes for which inhibitors were developed for clinical use; (ii) forCA I, II and IV, generally, aromatic sulfonamides behave as weakerinhibitors as compared to heterocyclic derivatives (compare 1-6, orDCP), as aromatic compounds, with 15, 21, AAZ, MZA, EZA, DZA or BRZamong others (as heterocyclic sulfonamides). In the case of CA IX, sucha fine distinction is rather difficult to be made, since both aromatic(such as 1, 6, 11, 12, 17, 18, 22-26) derivatives, as well asheterocyclic compounds (such as 14, 15, 21, and the clinically usedsulfonamides—except dichlorophenamide) possess rather similar inhibitionconstants, in the range of 14-50 nM; (iii) orthanilamide derivatives(such as 1, 17 and 22) behave as very potent CA IX inhibitors (K_(I)-sin the range of 20-33 nM), although they are weak or medium-weakinhibitors of CA I, II and IV; (iv) 1,3-benzene-disulfonamidederivatives (such as 11, 12 and DCP) are again strong CA IX inhibitors,with K_(I)-s in the range of 24-50 nM, although their CA II, I and IVinhibition profile is not particularly strong; (v) metanilamide 2,sulfanilamide 3, and 4-hydrazino-benzenesulfonamide 4 show CA IXinhibition data quite similar with those against CA II, whereashomosulfanilamide 5 and 4-aminoethyl-benzensulfonamide 6 act as betterCA IX inhibitors as compared to CA II inhibition; (vi) thehalogenosulfanilamides 7-10 are much weaker inhibitors of CA IX than ofCA II, a finding difficult to interpret at this moment; (vii) thestrongest CA II inhibitor among the investigated compounds,4-aminobenzolamide 15 (K_(I) of 2 nM) is not the strongest CA IXinhibitor (K_(I) of 38 nM). Instead, the best CA IX inhibitor detectedso far is the ethoxzolamide phenol 21 (K_(I) of 14 nM). It isinteresting to note that 21 and EZA have the same affinity for CA II,whereas their affinity for CA IX is rather different, with the phenolmore active than the ethoxy-derivative; (viii) among the clinically usedcompounds, the best inhibitor is acetazolamide, followed bymethazolamide, ethoxzolamide and brinzolamide. The most ineffective (butappreciably inhibiting the isozyme IX) are dichlorophenamide anddorzolamide; (ix) sulfonamides 20 and 22-26 behave as very good CA IXinhibitors, with K_(I)-s in the range of 16-32 nM, being slightly moreeffective than the clinically used CAIs mentioned above, and among thebest CA IX inhibitors detected so far. It is thus envisageable that suchcompounds may be used as lead molecules for obtaining more potent andeventually specific CA IX inhibitors, with applications as antitumoragents.

Screening of representative pyridinium derivatives of aromaticsulfonamides for inhibition of MN protein: From Example 2, whereinmembrane-impermeant pyridinium derivatives of sulfonamides were testedfor their ability to inhibit the enzymatic activity of CA IX, thefollowing conclusions were drawn from data of Table 2: (i) for a givensubstitution pattern of the pyridinium ring, the4-aminoethyl-benzenesulfonamide derivatives 55-70 were more active thanthe corresponding homosulfanilamide derivatives 39-54, which in turnwere more active than the corresponding sulfanilamides 27-38. Thisbehavior has also been observed for the other three investigatedisozymes [96]; (ii) some of the derivatives possessing bulkysubstitutents at the pyridinium ring (mainly phenyls, tert-butyls;n-butyl, n-propyl or iso-propyl), such as 34-37, 51 and 67, were veryineffective CA IX inhibitors, showing inhibition constants >500 nM;(iii) another group of compounds, including 27, 30-33, 44, and 60 showeda moderate inhibitory power towards the tumor-associated isozyme IX,showing K_(I) values in the range of 160-450 nM. Most of these compoundsare sulfanilamide derivatives (except 44 and 60), and the substitutionpattern at the pyridinium ring includes (with one exception, 27) atleast one phenyl group in 4, or two phenyls in the 2 and 4 positions. Itshould be noted that the corresponding homosulfanilamides and4-aminoethylbenzene-sulfonamides incorporating the same substitutionpattern as the compounds mentioned above (sulfanilamides), lead to muchbetter CA IX inhibitors (see later in the text); (iv) a third group ofderivatives, including 38, 45-50, 52, 53, 61, 63-66, 68 and 69, showedgood CA IX inhibitory properties, with K_(I) values in the range of64-135 nM. As mentioned above, except for thetetramethyl-pyridinium-substituted derivative 38, most of thesecompounds incorporate 4-phenyl-pyridinium or 2,4-diphenylpyridiniummoieties, whereas the group in position 6 is generally quite variable(alkyls or phenyl are tolerated). The most interesting observationregarding this subtype of CA IX inhibitors is constituted by the factthat the 2,4,6-triphenyl-pyridinium- and 2,6-diphenyl-pyridiniumderivatives of homosulfanilamide and 4-aminoethylbenzenesulfonamide(52-53 and 68-69) efficiently inhibit isozyme IX, although they act asvery weak inhibitors for isozymes I, II and IV (Table 2). As it will bediscussed shortly, this may be due to the fact that the hCA IX activesite is larger than that of the other investigated isozymes, notably CAII, I and IV; (v) a last group of derivatives (28-29; 39-43; 54; 55-59;62 and 70) showed very good CA IX inhibitory properties, these compoundspossessing K_(I) values in the range of 6-54 nM, similarly to theclinically used inhibitors acetazolamide, methazolamide,dichlorophenamide and indisulam, for which the inhibition data areprovided for comparison. It should be noted that three derivatives 58,59 and 70 showed inhibition constants <10 nM, these being the mostpotent CA IX inhibitors ever reported up to now. Correlated with theirmembrane-impermeability [96, 85], it may be assumed that in vivo suchcompounds may lead for the first time to a selective CA IX inhibition.Thus, the best substitution pattern at the pyridinium ring includeseither only compact alkyls (39-41, 54, 55 and 70), or2,6-dialkyl-4-phenyl-pyridinium moieties (all compounds mentioned aboveexcept 62, which incorporates a 2-methyl-4,6-diphenylpyridinium ring);(vi) the number of the substitutents at the pyridinium ring seems to beless important for the activity of this series of CAIs, since both di-,tri- or tetrasubstituted derivatives showed good inhibitory potency. Thenature of these groups on the other hand—as discussed in detail above—isthe most important parameter influencing CA inhibitory properties(together with the linker between the benzenesulfonamide moiety and thesubstituted pyridinium ring); (vii) the isozyme most similar to hCA IXregarding the affinity for these inhibitors was hCA II (which has 33%homology with hCA IX) [Pastorek et al. (1994), supra] whereas theaffinities of isozymes I and IV were rather different.

Screening of representative pyridinium derivatives of heterocyclicsulfonamides for inhibition of MN protein, and comparison withinhibition of other CA isozymes: Isozyme I. As seen from data of Table3, all derivatives 71-91 reported here act as very efficient CAIsagainst this isozyme which is generally the most “resistant” toinhibitors of this type [30, 31, 100, 102]. Indeed, aminobenzolamide isalready a highly potent CA I inhibitor (K_(I) of 6 nM), whereasinhibitors 71-91 show inhibition constants in the range of 3-12 nM, incontrast to the clinically used sulfonamide CAIs which are much lesseffective inhibitors, with K_(I) values in the range of 30-1200 nM(Table 3). Thus, derivatives possessing several bulky groups (i-Pr;t-Bu; n-Pr; n-Bu; Ph, etc) substituting the pyridinium moiety, such as73, 74, 77, 78, 82, 84, 85 showed a decreased inhibitory activity ascompared to aminobenzolamide, with K_(I) values in the range of 7-12 nM(aminobenzolamide has a K_(I) of 6 nM against hCA I). The rest of thecompounds were more efficient as compared to aminobenzolamide ininhibiting this isozyme, with K_(I) values in the range of 3-5 nM. BestCA I inhibitors were 75, and 89-91 (K_(I) of 3 nM), all of whichcontaining either only alkyl moieties or 4-Ph and other alkyl moietiessubstituting the pyridinium ring. These are probably the best CA Iinhibitors ever reported up to now, since the clinically used CAIs showmuch higher inhibition constants against isozyme I (Table 3).

Isozyme II. Aminobenzolamide is already a very potent CA II inhibitor,with an inhibition constant around 2 nM. Several of the new inhibitors,such as 74, 77, 78, 82-88 act as weaker CA II inhibitors as compared toaminobenzolamide, with K_(I) values in the range of 3.13-5.96 nM (butall these compounds act as potent inhibitors, being much more effectivethan the clinically used CAIs acetazolamide, methazolamide,dichlorophenamide or indisulam—see Table 3). Again the substitutionpattern at the pyridinium ring is the main discriminator of activity forthese compounds: all the less active derivatives mentioned aboveincorporate at least two bulky/long aliphatic groups, mainly inpositions 2- and 6- of the pyridinium ring (n-Pr; t-Bu; n-Bu; and Ph).The best CA II inhibitors among derivatives 71-91 were thoseincorporating more compact 2,6-substituents at the pyridinium ring (suchas Me, Et) together with a 4-Me or 4-Phe moiety, or those incorporatingonly aliphatic such groups, such as 71-73, 75, 76, 79-81, 89-91, whichshowed K_(I) values in the range of 0.20-1.61 nM (thus, for the bestinhibitors a factor of 10 increase in inhibitory power as compared toaminobenzolamide). It should be mentioned that iso-propyl-substitutedcompounds (73, 79) are active as CA II inhibitors, although theiractivity against CA I was not so good.

Isozyme IV. Most sulfonamides show inhibitory activity against CA IVintermediate between those towards CA I (less susceptible) and CA II(very high affinity for sulfonamides). This is also the trend observedwith the sulfonamides investigated here, derivatives ofaminobenzolamide. Thus, the parent sulfonamide (shown in FIG. 5) is apotent CA IV inhibitor, with a K_(I) value around 5 nM. The newderivatives of general formula (B) incorporating bulky pyridinium-ringsubstituents (such as 74, 77, 78, 82, 84-88, 90) were less effectivethan aminobenzolamide, showing K_(I) values in the range of 5.2-10.3 nM,whereas the compounds showing the other substitution pattern mentionedabove were better CA IV inhibitors, showing K_(I) values in the range of2.0-4.7 nM.

Isozyme IX. Aminobenzolamide is less inhibitory against this isozyme(K_(I) of 38 nM) as compared to other isozymes discussed above. Thisbehavior is difficult to explain at this point, since no X-ray crystalstructure of this isozyme has been reported. A very encouraging resultobtained with the new derivatives of general formula (B) reported here,was the observation that several of them show very high affinity for CAIX, with K_(I) values in the range of 3-9 nM (derivatives 71, 72, 75,76, and 89). It may be seen that all of them incorporate aliphaticmoieties (Me, Et and i-Pr) in positions 2- and 6- of the pyridiniumring, and either 4-Me or 4-Ph moieties. Only one compound istetrasubstituted (89), again possessing only methyl moieties. The bestCA IX inhibitor (and the best ever reported up to now) was 71, which isalmost 13 times more effective than benzolamide in inhibiting thisisozyme. Another group of new derivatives, such as 73, 74, 77, 79, 80,81, 83, 86-88, 90, 91, showed effective CA IX inhibition, with K_(I)values in the range of 12-35 nM, being thus more effective thanaminobenzolamide. They incorporate slightly bulkier groups as comparedto the previously discussed ones. Again the less effective inhibitors(K_(I) values in the range of 40-43 nM) were those incorporating severalbulky pyridinium substituents, such as 78, 84, 85 which contained eithertwo n-Bu or one Ph and n-Bu/t-Bu in positions 2- and 6- of thepyridinium ring. Thus, SAR is now rather clear for this type of CAIs:best CA IX inhibitors should contain either only small, compactaliphatic moieties substituting the pyridinium ring, or they tolerate a4-Ph moiety, but the 2,6-substituents should again be small, compactaliphatic moieties. In this particular case,2,4,6-trisubstituted-pyridinium derivatives were more effective CA IXinhibitors as compared to the tetrasubstituted derivatives.

Membrane impermeability of Heterocyclic Sulfonamide Inhibitors of CA IX.As seen from data of Table 4 of Example 3, incubation of human red cells(which contain high concentrations of isozymes I and II, i.e., 150 μMhCA I and 20 μM hCA II, but not the membrane-bound CA IV or CA IX) [118]with millimolar concentrations of different sulfonamide inhibitors, suchas acetazolamide, or methazolamide, led to saturation of the twoisozymes present in erythrocytes with inhibitor, already after shortperiods of incubation (30 min), whereas for benzolamide oraminobenzolamide, a similar effect is achieved after somehow longerperiods (60 min) (Table 4). This is obviously due to the highdiffusibility through membranes of the first three inhibitors, whereasbenzolamide/aminobenzolamide with a pK_(a) of 3.2 for the secondsulfonamido group [58] being present mainly as an (di)anion at the pH atwhich the experiment has been done (7.4), is already less diffusible andpenetrates membranes in a longer time. Different cationic sulfonamidessynthesized by us here, such as 71, 76, 89, 91, in the same conditions,were detected only in very small amounts within the blood red cells,proving that they were unable to penetrate through the membranes,obviously due to their cationic nature. Even after incubation times aslong as one hour (and longer, data not shown), only traces of suchcationic sulfonamides were present inside the blood red cells, as provedby the three assay methods used for their identification in the celllysate, which were in good agreement with each other (Table 4). Thisdemonstrates that the proposed approach for achieving membraneimpermeability works well for the designed positively-chargedsulfonamide CAIs of the general formula (B) (shown above), since thevery small amount of sulfonamide detected may be due to contamination ofthe lysates with very small amount of membranes.

Design of Membrane-Impermeant Sulfonamide Inhibitors of CA IX

No X-ray crystal structure of isozyme IX is available up to now, instrong contrast with hCA II, for which many X-ray crystal structures areavailable (alone or in complexes with inhibitors and activators) [1, 2,14, 15, 19a, 19b, 37, 38]. Examining the active site residues of thesetwo isozymes and the architecture of hCA II, may help explain the aboveinhibition data and their relevance for CA IX specific inhibitors.

First of all, the zinc ligands and the proton shuttle residue of thesetwo isozymes are identical [33, 43, 72, 100, 101, 102, 114, 115, 117].An important difference is constituted by the amino acid in position131, which is Phe for hCA II and Val for hCA IX. Phe 131 is known to bevery important for the binding of sulfonamide inhibitors to hCA II [2,46, 47]: in many cases this bulky side chain limits the space availablefor the inhibitor aromatic moieties, or it may participate in stackinginteractions with groups present in it (for recent examples see refs.[2, 46, 47]. Thus, the presence of a less bulky such residue in hCA IX(i.e., a valine) which is also unavailable for participation to stackinginteractions has as a consequence the fact that the hCA IX active siteis larger than that of hCA II. A second potentially important residue is132, which is Gly in hCA II and Asp in hCA IX. This residue is situatedon the rim of the hydrophilic half of the entrance to the active site ofhCA II (and presumably also of hCA IX) and it is critical for theinteraction with inhibitors possessing elongated molecules, as recentlyshown by us [19b]. Strong hydrogen bonds involving the CONH moiety ofGly 132 were shown to stabilize the complex of this isozyme with ap-aminoethylbenzenesulfonamide derived inhibitor [19b]. In the case ofhCA IX, the presence of aspartic acid in this position at the entranceof the active site may signify that: (i) stronger interactions withpolar moieties of the inhibitor bound within the active site should bepossible, since the COOH moiety possesses more donor atoms; (ii) thisresidue may have flexible conformations, fine-tuning in this way theinteraction with inhibitors. Thus, the stronger hCA IX inhibition withsome of these inhibitors (as compared to their affinity for isozyme II),such as for example 46-50, 52, 53, 55, 58, 62 and 68-70, might beexplained just by the different interactions with the two active siteresidues mentioned above.

Therapeutic Use of MN-Specific Inhibitors

The MN-specific inhibitors of this invention, organic and/or inorganic,preferably organic, and as outlined above, may be used therapeuticallyin the treatment of neoplastic and/or pre-neoplastic disease, eitheralone or in combination with other chemotherapeutic drugs.

The MN-specific inhibitors can be administered in a therapeuticallyeffective amount, preferably dispersed in a physiologically acceptable,non-toxic liquid vehicle.

The MN-specific inhibitors used according to the methods of theinvention may exploit activation of CA IX under hypoxia, to specificallytarget hypoxic conditions, acidic conditions or both conditions.

In addition to targeting hypoxia, CA IX selective inhibitors may be usedtherapeutically to increase pHe in order to reduce tumor aggressivenessand drug uptake. It is known that the atypical pH gradient of tumorcells (acidic extracellular pH, neutral-to-basic intracellular pH) actsto exclude weak base drugs such as the anthracyclines and vincaalkaloids. In two different mouse tumor models, alkalinization of tumorextracellular pH (using bicarbonate pretreatment) enhanced theanti-tumor activity of the weak base chemotherapeutic agents doxorubicinand mitoxantrone [126, 137]. Most combination chemotherapy regimensinclude at least one weak base drug, and it may be possible to enhancethe efficacy of such drugs with the co-administration of CA IX-specificinhibitors.

Diagnostic/Prognostic and Therapeutic Use of MN-Specific InhibitorsWhich Selectively Bind Activated CA Domain of CA IX

As used herein, “normoxia” is defined as oxygen tension levels in aspecific vertebrate tissue that are within the normal ranges ofphysiological oxygen tension levels for that tissue. As used herein,“hypoxia” is defined as an oxygen tension level necessary to stabilizeHIF-1α in a specific tissue or cell. Experimentally-induced hypoxia isgenerally in the range of 2% pO₂ or below, but above anoxia (0% pO₂, asanoxia would be lethal). The examples described herein that concernhypoxia were performed at 2% pO₂ which is an exemplary hypoxiccondition. However, ones of skill in the art would expect other oxygentension levels to be understood as “hypoxic” and to produce similarexperimental results. For example, Wykoff et al. [121] used a conditionof 0.1% pO₂ as representative of hypoxia to induce HIF-1α-dependentexpression of CA9. Tomes et al. has demonstrated varying degrees ofHIF-1α stabilization and CA9 expression in HeLa cells or primary humanbreast fibroblasts under exemplary in vitro hypoxic conditions of 0.3%,0.5% and 2.5% pO₂ [Tomes et al., Br. Cancer Res. Treat., 81(1): 61-69(2003)]. Alternatively, Kaluz et al. has used the exemplary hypoxiccondition of 0.5% pO₂ for experimental induction of CA9 [Kaluz et al.,Cancer Res., 63: 917-922 (2003)] and referred to “experimentally-inducedranges” of hypoxia as 0.1-1% pO₂ [129].

Oxygen tension levels above 2% pO₂ may also be hypoxic, as shown byTomes et al., supra. One of skill in the art would be able to determinewhether a condition is hypoxic as defined herein, based on adetermination of HIF-1α stabilization. Exemplary ranges of hypoxia in aspecific tissue or cell may be, for example, between about 3% to about0.05% pO₂, between about 2% to about 0.1% pO₂, between about 1% to about0.1% pO₂, and between about 0.5% to about 0.1% pO₂.

“Mild hypoxia” is defined herein as an oxygen tension level in aspecific vertebrate tissue that does not stabilize HIF-1α and is belownormoxia. Mild hypoxia would be understood by those of skill in the artto be below normoxia but still above hypoxia.

As has been previously reported by the inventors and others, CA IX canbe expressed by alternative mechanisms, at least in vitro: ahypoxia-regulated pathway which requires HIF-1α stabilization, and aphosphatidylinositol 3′ kinase (PI3K) pathway occurring at high celldensity, which requires a minimal level of HIF-1α and a lowered oxygenconcentration that is, however, above that necessary for HIF-1αstabilization [129]. Cell crowding may lead to pericellular mildhypoxia; for example, in dense LNCaP human prostate carcinoma cells 48hours after plating, oxygen tension was 9% pO₂ above the cell surface,compared with 13% pO₂ above sparse cells, and compared with 0.1-1% pO₂frequently used in experimentally induced hypoxic responses [Sheta etal., Oncogene, 20: 7624-7634 (2001)]. The cell density-dependentinduction of CA IX may explain CA IX expression in areas adjacent tohypoxic regions in solid tumors, and the selectivity of the CAIX-specific inhibitors for hypoxically-induced CA IX may be exploited todifferentiate between the two mechanisms diagnostically, prognosticallyand therapeutically.

The CA IX-specific inhibitors which selectively bind the activated formof CA IX can be used, for example, in laboratory diagnostics, usingfluorescence microscopy or histochemical staining; as a component inassays for detecting and/or quantitating MN antigen in, for example,clinical samples; in electron microscopy with colloid gold beads forlocalization of MN proteins and/or polypeptides in cells; and in geneticengineering for cloning the MN gene or fragments thereof, or relatedcDNA. Such activated CA IX-specific inhibitors can be used as componentsof diagnostic/prognostic kits, for example, for in vitro use onhistological sections; such inhibitors can be labeled appropriately, aswith a suitable radioactive isotope, and used in vivo to locatemetastases by scintigraphy. Further such inhibitors may be used in vivotherapeutically to treat cancer patients with or without toxic and/orcytostatic agents attached thereto. Further, such inhibitors can be usedin vivo to detect the presence of neoplastic and/or pre-neoplasticdisease. Still further, such inhibitors can be used to affinity purifyMN proteins and polypeptides.

Such CA IX-specific inhibitors which selectively bind activated CA IXcould be used in combination with other compounds which bind to anyforms of CA IX, in order to differentiate between hypoxic and nonhypoxicexpression of CA IX. For example, such methods could comprise theimmunohistochemical use of a CA IX-specific sulfonamide, such ascompound 92, and an antibody which binds to the PG domain of CA IX, suchas the M75 Mab. If a tissue overexpresses CA IX, as indicated by M75 Mabbinding, but such CA IX is not activated, as indicated by lack of CAIsulfonamide binding, it would indicate that the CA IX expression isinduced by a nonhypoxic condition which elevates CA IX levels, such asby cell density-dependent induction of CA IX mediated byphosphatidylinositol 3′-kinase (PI3K). Alternatively, the methods of theinvention could comprise the use of an antibody which specifically bindsthe activated form of the CA domain of CA IX, in combination with anantibody which binds to another domain of CA IX, such as the Mab M75which binds to the PG domain, in order to differentiate between hypoxicand nonhypoxic expression of CA IX.

Such information may be useful as a method of indicating degrees oflowered oxygen tension; for example, at intermediate oxygen tensionlevels (for example, such as between 9% and 5% pO₂), CA IX may beinduced, but not activated, and may be detected only by the MAb M75;whereas at hypoxic pO₂ levels (such as 2% or less), CA IXprotein/polypeptide may be both expressed and activated, and detectableby both the Mab M75 and by a CAI sulfonamide which specifically bindsthe activated CA domain of CA IX. Thus, detection of both the presenceof CA IX and its specific binding by CA IX-specific CAIs can be used incombination as a noninvasive method to determine pO₂ levels of a tissue.Such information may be useful diagnostically/prognostically or inpatient therapy selection, depending upon which CA IX functions aretargeted. For example, in in vitro RNA interference studies, expressionof CA IX under both normoxia and hypoxia promoted tumor growth, inadditive effects [138]. Those data along with the present inventionimplicate more than one CA IX function as promoting tumor growth.Radiobiologically relevant tumor hypoxia appears to occur at loweroxygen tension levels, such as at 2% pO₂ or lower, which oxygen tensionlevels may induce activated CA IX expression that is detectable by CAIX-specific inhibitors. It is possible that tumors that express CA IXconstitutively because of deregulation [such as deregulated PI3Kactivity; 129] may be distinguished by the CA IX being detectable by theMab M75 but not by CA IX-specific inhibitors (because the CA domain isoverexpressed but not activated).

Materials and Methods

General. Melting points: heating plate microscope (not corrected); IRspectra: KBr pellets, 400-4000 cm⁻¹ Perkin-Elmer 16PC FTIR spectrometer;¹H-NMR spectra: Varian 300CXP apparatus (chemical shifts are expressedas δ values relative to Me₄Si as standard); Elemental analysis: CarloErba Instrument CHNS Elemental Analyzer, Model 1106. All reactions weremonitored by thin-layer chromatography (TLC) using 0.25-mm precoatedsilica gel plates (E. Merck). Pyrylium salts were prepared by literatureprocedures, generally by olefin (or their precursors) bisacylation, asdescribed in the literature [6, 26, 108], whereas aminobenzolamide asdescribed earlier [97]. Other sulfonamides used as standards werecommercially available.

General Procedure for the Preparation of Compounds 71-91 (PyridiniumDerivatives of Aminobenzolamide)

An amount of 2.9 mM of aminobenzolamide [97] and 2.9 mM of pyrylium saltII (depicted in FIG. 5) were suspended in 5 mL of anhydrous methanol andpoured into a stirred mixture of 14.5 mM of triethylamine and 5.8 mM ofacetic anhydride. After five minutes of stirring, another 10 mL ofmethanol were added to the reaction mixture, which was heated to refluxfor 15 min. Then 14.5 mM of acetic acid was added and heating wascontinued for 2-5 hours. The role of the acetic anhydride is to reactwith the water formed during the condensation reaction between thepyrylium salt and the aromatic amine, in order to shift the equilibriumtowards the formation of the pyridinium salts of the general formula (B)(shown above). In the case of aminobenzolamide, this procedure is theonly one which gave acceptable yields in pyridinium salts, probably dueto the deactivating effect of the sulfamoylaminothiadiazole moiety onthe amine group, which becomes poorly nucleophilic and unreactivetowards these reagents. The precipitated pyridinium salts obtained werepurified by treatment with concentrated ammonia solution (which alsoconverts the eventually unreacted pyrylium salt to the correspondingpyridine which is soluble in acidic medium), reprecipitation withperchloric acid and recrystallization from water with 2-5% HClO₄.

Purification of Catalytic Domain of CA IX

The cDNA of the catalytic domain of hCA IX (isolated as described byPastorek et al. [72]) was amplified by using PCR and specific primersfor the vector pCAL-n-FLAG (from Stratagene). The obtained construct wasinserted in the pCAL-n-FLAG vector and then cloned and expressed inEscherichia coli strain BL21-GOLD(DE3) (from Stratagene). The bacterialcells were lysed and homogenated in a buffered solution (pH 8) of 4 Murea and 2% Triton X-100, as described by Wingo et al. [116]. Thehomogenate thus obtained was extensively centrifuged in order to removesoluble and membrane associated proteins as well as other cellulardebris. The resulting pellet was washed by repeated homogenation andcentrifugation in water, in order to remove the remaining urea andTriton X-100. Purified CA IX inclusion bodies were denaturated in 6 Mguanidine hydrochloride and refolded into the active form by snapdilution into a solution of 100 mM MES (pH 6), 500 mM L-arginine, 2 mMZnCl₂, 2 mM EDTA, 2 mM reduced glutathione, 1 mM oxidized glutathione.Active hCA IX was extensively dialysed into a solution of 10 mM Hepes(pH 7.5), 10 mM Tris HCl, 100 mM Na₂SO₄ and 1 mM ZnCl₂. The amount ofprotein was determined by spectrophometric measurements and its activityby stopped-flow measurements, with CO₂ as substrate [44]. Optionally,the protein was further purified by sulfonamide affinity chromatography[44], the amount of enzyme was determined by spectrophometricmeasurements and its activity by stopped-flow measurements, with CO₂ assubstrate [44].

CA I, II and IV Purification

Human CA I and CA II cDNAs were expressed in Escherichia coli strainBL21 (DE3) from the plasmids pACA/hCA I and pACA/hCA II described byLindskog's group [54]. Cell growth conditions were those described inref. [12], and enzymes were purified by affinity chromatographyaccording to the method of Khalifah et al. [45]. Enzyme concentrationswere determined spectrophotometrically at 280 nm, utilizing a molarabsorptivity of 49 mM⁻¹.cm⁻¹ for CA I and 54 mM⁻¹.cm⁻¹ for CA II,respectively, based on M_(r)=28.85 kDa for CA I, and 29.3 kDa for CA II,respectively [53, 84]. CA IV was isolated from bovine lung microsomes asdescribed by Maren et al, and its concentration has been determined bytitration with ethoxzolamide [59].

Enzyme Assays

CA CO₂ Hydrase Activity Assay

An SX.18MV-R Applied Photophysics stopped-flow instrument has been usedfor assaying the CA CO₂ hydration activity assays [44]. A stopped flowvariant of the Poker and Stone spectrophotometric method [76] has beenemployed, using an SX.18MV-R Applied Photophysics stopped flowinstrument, as described previously [43]. Phenol red (at a concentrationof 0.2 mM) has been used as indicator, working at the absorbance maximumof 557 nm, with 10 mM Hepes (pH 7.5) as buffer, 0.1 M Na₂SO₄ (formaintaining constant the ionic strength), following the CA-catalyzed CO₂hydration reaction for a period of 10-100 s. Saturated CO₂ solutions inwater at 20° C. were used as substrate [44]. Stock solutions ofinhibitor (1 mM) were prepared in distilled-deionized water with 10-20%(v/v) DMSO (which is not inhibitory at these concentrations) anddilutions up to 0.01 nM were done thereafter with distilled-deionizedwater. Inhibitor and enzyme solutions were preincubated together for 10min at room temperature prior to assay, in order to allow for theformation of the E-I complex. Triplicate experiments were done for eachinhibitor concentration, and the values reported throughout the paperare the mean of such results.

CA Esterase Activity Assay

Initial rates of 4-nitrophenylacetate hydrolysis catalysed by differentCA isozymes were monitored spectrophotometrically, at 400 nm, with aCary 3 instrument interfaced with an IBM compatible PC [76]. Solutionsof substrate were prepared in anhydrous acetonitrile; the substrateconcentrations varied between 2.10⁻² and 1.10⁻⁶ M, working at 25° C. Amolar absorption coefficient ε of 18,400 M⁻¹.cm⁻¹ was used for the4-nitrophenolate formed by hydrolysis, in the conditions of theexperiments (pH 7.40), as reported in the literature [76]. Non-enzymatichydrolysis rates were always subtracted from the observed rates.Triplicate experiments were done for each inhibitor concentration, andthe values reported throughout the paper are the mean of such results.Stock solutions of inhibitor (1-3 mM) were prepared indistilled-deionized water with 10-20% (v/v) DMSO (which is notinhibitory at these concentrations) and dilutions up to 0.01 nM weredone thereafter with distilled-deionized water. Inhibitor and enzymesolutions were preincubated together for 10 min at room temperatureprior to assay, in order to allow for the formation of the E-I complex.The inhibition constant K_(I) was determined as described in references[44, 76].

Membrane Permeance Assay: Ex Vivo Penetration Through Red Blood Cells

An amount of 10 mL of freshly isolated human red cells thoroughly washedseveral times with Tris buffer (pH 7.40, 5 mM) and centrifuged for 10min were treated with 25 mL of a 2 mM solution of sulfonamide inhibitor.Incubation has been done at 37° C. with gentle stirring, for periods of30-120 min. After the incubation times of 30, 60 and 120 min.,respectively, the red cells were centrifuged again for 10 min, thesupernatant discarded, and the cells washed three times with 10 mL ofthe above mentioned buffer, in order to eliminate all unbound inhibitor[81, 96, 98]. The cells were then lysed in 25 mL of distilled water,centrifuged for eliminating membranes and other insoluble impurities.The obtained solution was heated at 100° C. for 5 minutes (in order todenature CA-s) and sulfonamides possibly present have been assayed ineach sample by three methods: a HPLC method [36]; spectrophotometrically[4] and enzymatically [76].

HPLC: A variant of the methods of Gomaa [36] has been developed by us,as follows: a commercially available 5 μm Bondapak C-18 column was usedfor the separation, with a mobile phase made ofacetonitrile—methanol—phosphate buffer (pH 7.4) 10:2:88 (v/v/v), at aflow rate of 3 mL/min, with 0.3 mg/mL sulphadiazine (Sigma) as internalstandard. The retention times were: 12.69 min for acetazolamide; 4.55min for sulphadiazine; 10.54 min for benzolamide; 12.32 min foraminobenzolamide; 3.15 min for 71; 4.41 min for 76; 3.54 min for 89; and4.24 min for 91. The eluent was monitored continuously for absorbance(at 254 nm for acetazolamide, and wavelength in the range of 270-310 nmin the case of the other sulfonamides.

Spectrophotometrically: A variant of the pH-induced spectrophotometricassay of Abdine et al. [4] has been used, working for instance at 260and 292 nm, respectively, for acetazolamide; at 225 and 265 nm,respectively, for sulfanilamide, etc. Standardized solutions of eachinhibitor have been prepared in the same buffer as the one used for themembrane penetrability experiments.

Enzymatically: the amount of sulfonamide present in the lysate has beenevaluated based on hCA II inhibition measured with the esterase method,as described above [76]. Standard inhibition curves have been obtainedpreviously for each sulfonamide, using the pure compound, which wereused thereafter for determining the amount of inhibitor present in thelysate. Mention should be made that the three methods presented aboveled to results in good agreement, within the limits of the experimentalerrors.

Statistical analysis: Values are expressed ±standard error ofmeasurement. Statistical significance was determined using an unpairedt-test with p<0.05 considered significant.

The following materials and methods were used for Examples 4-8.

Cell Culture

MDCK, SiHa, HeLa cells and their transfected derivatives were grown inDMEM with 10% FCS and buffered with 22.3 mM bicarbonate [103]. Tomaintain the standard conditions, the cells were always plated in 3 mlof culture medium at a density of 0.8-1×10⁶ per 6 cm dish 24 hoursbefore the transfer to hypoxia (2% O₂ and 5% CO₂ balanced with N₂)generated in a Napco 7000 incubator. Parallel normoxic dishes wereincubated in air with 5% CO₂. At the end of each experiment, the pH ofthe culture medium was immediately measured, the medium was harvestedfor the determination of the lactic acid content with the standard assaykit (Sigma), the cells were counted to ensure that the resultingcultures were comparable, and then processed either forimmunofluorescence or extracted for immunoprecipitation and/orimmunoblotting.

Sulfonamide Synthesis and Treatment of Cells

Compound 6 sulfonamide [4-(2-aminoethyl)-benzenesulfonamide] was fromSigma-Aldrich. The membrane-impermeable Compound 39[4-(2,4,6-trimethylpyridinium-N-methylcarboxamido)-benzenesulfonamideperchlorate] was prepared by reaction of homosulfanilamide with2,4,6-trimethylpyrilium perchlorate [81]. Compound 92 [the fluorescentderivative of Compound 5 sulfonamide] was obtained fromhomosulfanilamide and fluorescein isothiocyanate [142]. CAIs showed thefollowing K_(I) values assessed by CO₂ hydration methods using thepurified CA domain of CA IX: Compound 636 nM, Compound 3938 nM andCompound 92 24 nM. The sulfonamides were dissolved in PBS with 20% DMSOat 100 mM concentration and diluted in a culture medium to a requiredfinal concentration just before their addition to cells. The cells wereincubated for 48 hours in hypoxia and normoxia, respectively, the pH ofthe culture medium was measured and the binding of the FITC-labeledCompound 92 to living cells, washed three times with PBS, was viewed bya Nikon E400 epifluorescence microscope.

Cloning of CA IX Mutants and Transfection

Cloning of CA IX deletion mutants lacking either the N-terminal PGdomain or the central CA domain was performed as described [73, 145].MDCK and HeLa cell lines constitutively expressing CA IX protein or itsmutants were obtained by cotransfection of recombinant plasmids pSG5C-CAIX, pSG5C-ΔCA and pSG5C-ΔPG with pSV2neo plasmid in 10:1 ratio using aGenePorter II transfection kit from Gene Therapy Systems. Thetransfected cells were subjected to selection in 500-1000 μg/ml G418,cloned, tested for CA IX and expanded. At least three clonal cell linesexpressing each CA IX form were analyzed to eliminate the effect ofclonal variation. The cells cotransfected with empty pSG5C and pSV2 neowere used as negative controls.

Indirect Immunofluorescence and Immunoblotting

Cells grown on glass coverslips were fixed in ice-cold methanol at −20°C. for 5 min and stained with CA IX-specific MAb M75 directed to the PGdomain or V/10 directed to the CA domain followed by FITC-labeledsecondary antibodies [73, 145]. For immunoblotting, cells were rinsedwith PBS and extracted in RIPA buffer for 30 min on ice. Proteinconcentrations were quantified using the BCA kit (Pierce). The proteins(50 μg/lane) were resolved in 10% SDS-PAGE under reducing andnon-reducing conditions, respectively, transferred to PVDF membrane andCA IX was detected with the specific MAbs as described [73].

Cell Biotinylation and Immunoprecipitation

Cells were washed with ice-cold buffer A (20 mM sodium hydrogencarbonate, 0.15 M NaCl, pH 8.0), incubated for 60 min at 4° C. withbuffer A containing 1 mg of NHS-LC-Biotin (Pierce), then washed 5 timeswith buffer A and extracted in RIPA as described above. MAb V/10(deposited at the BCCM™/LMBP Plasmid Collection Laboratorium, Gent,Belgium under Accession No. LMBP 6009CB) in 1 ml of hybridoma medium wasbound to 25 μl 50% suspension of Protein-A Sepharose (Pharmacia) for 2 hat RT. Biotinylated extract (200 μl) was pre-cleared with 20 μl of 50%suspension of Protein-A Sepharose and then added to the bound MAb.Immunocomplexes collected on the Protein-A Sepharose were separated bySDS-PAGE, transferred to a PVDF membrane and revealed withperoxidase-conjugated streptavidin ( 1/1000, Pierce) followed byenhanced chemoluminiscence.

The following examples are for purposes of illustration only and are notmeant to limit the invention in any way.

Example 1 Inhibition of the Tumor-Associated Isozyme IX with Aromaticand Heterocyclic Sulfonamides

The inhibition of the tumor-associated transmembrane carbonic anhydraseIX (CA IX) isozyme has been investigated with a series of aromatic andheterocyclic sulfonamides, including the six clinically used derivativesacetazolamide, methazolamide, ethoxzolamide, dichlorophenamide,dorzolamide and brinzolamide. Inhibition data for the physiologicallyrelevant isozymes I and II (cytosolic forms) and IV (membrane-bound)were also provided for comparison.

Chemistry. Sulfonamides investigated for the inhibition of thetumor-associated isozyme CA IX, of types 1-26 are shown in FIG. 4A-B.Compounds 1-6, 11-12, 20 and 26 are commercially available, whereas 7-10[43], 13-19 [24, 79, 90, 97] and 21-25 [79] were prepared as reportedearlier. The six clinically used compounds were also assayed, since nosuch data are available in the literature.

CA inhibition data. Inhibition data against four CA isozymes, CA I, II,IV and IX [44, 72, 116], with the above mentioned compounds I-26 and thesix clinically used inhibitors, are shown in Table 1.

TABLE 1 CA I, II, IV and IX inhibition data with sulfonamides 1-26 andclinically used inhibitors. K_(I)* (nM) Inhibitor hAC I^(a) hCA II^(a)bCA IV^(b) hCA IX^(c)  1 45400 295 1310 33  2 25000 240 2200 238  328000 300 3000 294  4 78500 320 3215 305  5 25000 170 2800 103  6 21000160 2450 33  7 8300 60 180 245  8 9800 110 320 264  9 6500 40 66 269 106000 70 125 285 11 5800 63 154 24 12 8400 75 160 39 13 8600 60 540 41 149300 19 355 30 15 6 2 5 38 16 164 46 129 34 17 185 50 144 20 18 109 3372 31 19 95 30 72 24 20 690 12 154 16 21 55 8 17 14 22 21000 125 415 3223 23000 133 438 30 24 24000 125 560 21 25 18000 110 450 22 26 135 40 8626 AAZ 250 12 70 25 MZA 50 14 36 27 EZA 25 8 13 34 DCP 1200 38 380 50DZA 50000 9 43 52 BRZ — 3 45 37 ^(a)Human cloned isozymes, esteraseassay method [76]; ^(b)Isolated from bovine lung microsomes, esteraseassay method [76]; ^(c)Human cloned isozyme, CO₂ hydrase assay method[44, 72, 116].

We report here the first inhibition study of the tumor-associated,transmembrane isozyme CA IX with a series of aromatic and heterocyclicsulfonamides, including also the six clinically used derivativesacetazolamide, methazolamide, ethoxzolamide, dichlorophenamide,dorzolamide and brinzolamide. Inhibition data for the physiologicallyrelevant isozymes I and II (cytosolic forms) and IV (membrane-bound) arealso provided for comparison. Very interesting inhibition profileagainst CA IX with these sulfonamides has been detected, which is apromising discovery for the potential design of CA IX-specificinhibitors, with applications as antitumor agents. Several nanomolar CAIX inhibitors have been detected, both among the aromatic (such asorthanilamide, homosulfanilamide, 4-carboxy-benzenesulfonamide,1-naphthalene-sulfonamide and 1,3-benzenedisulfonamide derivatives) aswell as the heterocyclic (such as 1,3,4-thiadiazole-2-sulfonamide,benzothiazole-2-sulfonamide, etc.) sulfonamides investigated.

Example 2 The First Selective, Membrane-Impermeant Inhibitors Targetingthe Tumor-Associated Isozyme IX

Up to now no CA IX inhibition studies with this type ofmembrane-impermeant CAIs have been reported. Thus, we decided to exploresome of the pyridinium derivatives of general formula (A) for theirinteraction with the catalytic domain of tumor-associated isozyme IX,recently cloned and purified by the inventors [33, 43, 114, 115, 117],as well as the cytosolic, physiologically relevant isozymes CA I, II andthe membrane-anchored isozyme CA IV [88, 96].

The inhibition of the tumor-associated transmembrane carbonic anhydraseIX (CA IX) isozyme has been investigated with a series ofpositively-charged, pyridinium derivatives of sulfanilamide,homosulfanilamide and 4-aminoethyl-benzenesulfonamide. Inhibition datafor the physiologically relevant isozymes I and II (cytosolic forms) andIV (membrane-bound) were also provided for comparison. This is the firstreport of inhibitors that may selectively target CA IX, due to theirmembrane-impermeability and high affinity for this clinically relevantisozyme.

CA Inhibition

Data of Table 2 clearly show that most of the compounds 27-70 act asefficient CA IX inhibitors, and that their affinity for this isozymediffers considerably as compared to affinities for the cytosolicisozymes CA I and II, and the other membrane-associated isozymeinvestigated, CA IV.

In a series of substituted-pyridinium derived sulfanilamides,homosulfanilamides and p-aminoethylbenzenesulfonamides, a large numberof effective hCA IX inhibitors were detected. Some low nanomolar CA IXinhibitors were reported for the first time. Since these compounds aremembrane-impermeant due to their salt-like character, and as hCA IX ispresent on the extracellular side of many tumors with poor clinicalprognosis, compounds of this type target specifically thistumor-associate CA isozyme without affecting the cytosolic CAs known toplay important physiological functions. Thus, compounds of this type mayconstitute the basis of new anticancer therapies based on CA inhibitors.

TABLE 2 Inhibition of isozymes hCA I, hCA II, bCA IV and hCA IX with thepyridinium salts 27-70.

K_(I)* hCA I^(a) hCA II^(a) bCA IV^(b) hCA IX^(c) Compound R² R³ R⁴ R⁶(μM) (nM) (nM) (nM) 27 Me H Me Me 10 150 290 165 28 Me H Ph Me 7 60 21148 29 Et H Ph Et 6 60 182 43 30 n-Pr H Ph n-Pr 10 120 194 178 31 i-Pr HPh i-Pr 5 50 90 160 32 Me H Ph Ph 40 210 852 280 33 Et H Ph Ph 43 4001300 450 34 n-Pr H Ph Ph 140 580 1483 >500 35 i-Pr H Ph Ph 125 4402102 >500 36 n-Bu H Ph Ph 305 620 2155 >500 37 Ph H Ph Ph 290 5102500 >500 38 Me Me Me Me 5 40 61 72 39 Me H Me Me 7 50 92 38 40 i-Pr HMe Me 6 50 80 42 41 i-Pr H Me i-Pr 11 80 144 54 42 Me H Ph Me 4 20 70 2643 Et H Ph Et 2 21 52 29 44 n-Pr H Ph n-Pr 24 90 163 230 45 i-Pr H Phi-Pr 12 61 101 100 46 Me H Ph Ph 32 121 161 64 47 Et H Ph Ph 42 314 98379 48 n-Pr H Ph Ph 130 390 1260 85 49 i-Pr H Ph Ph 112 370 1214 80 50n-Bu H Ph Ph 300 595 2104 135 51 t-Bu H Ph Ph 110 321 1070 >500 52 Ph HPh Ph 280 472 1956 120 53 Ph H H Ph 280 493 1954 106 54 Me Me Me Me 3 3051 35 55 Me H Me Me 4 21 60 14 56 i-Pr H Me Me 2 15 32 31 57 i-Pr H Mei-Pr 3 20 70 49 58 Me H Ph Me 1 8 20 6 59 Et H Ph Et 1 9 21 8 60 n-Pr HPh n-Pr 7 42 82 205 61 i-Pr H Ph i-Pr 6 21 70 89 62 Me H Ph Ph 18 103144 37 63 Et H Ph Ph 40 220 761 70 64 n-Pr H Ph Ph 112 270 1055 84 65i-Pr H Ph Ph 94 350 864 78 66 n-Bu H Ph Ph 290 544 2008 120 67 t-Bu H PhPh 92 275 1000 >500 68 Ph H Ph Ph 270 419 1830 95 69 Ph H H Ph 265 4201905 81 70 Me Me Me Me 2 10 21 8 acetazolamide 0.25 12 70 25methazolamide 0.05 14 36 27 dichlorophenamide 1.2 38 380 50 indisulam0.03 15 65 24 ^(a)Human (cloned) isozymes; ^(b)From bovine lungmicrosomes; ^(c)Catalytic domain of the human, cloned isozyme. *errorsin the range of ± 10% of the reported value, from three differentdeterminations. For compunds 27-38: n = 0; 39-54: n = 1; 55-70: n = 2

Example 3 Design of Selective, Membrane-Impermeant HeterocyclicSulphonamide Inhibitors Targeting the Human Tumor-Associated Isozyme IX

A series of positively-charged sulfonamides were obtained by reaction ofaminobenzolamide(5-(4-aminobenzenesulfonylamino)-1,3,4-thiadiazole-2-sulfonamide) withtri-/tetra-substituted pyrilium salts possessing alkyl-, aryl- orcombinations of alkyl and aryl groups at the pyridinium ring. These newcompounds are membrane-impermeant due to their salt-like character andwere assayed for the inhibition of four physiologically relevantcarbonic anhydrase (CA, EC 4.2.1.1) isozymes, the cytosolic hCA I andII, the membrane-anchored bCA IV and the membrane-bound, tumorassociated isozyme hCA IX. The high affinity of these new derivativesfor the tumor-associated isozyme CA IX and their membraneimpermeability, make this type of CA inhibitors interesting candidatesfor the selective inhibition of only the tumor associated isozyme andnot the cytosolic ones, for which they also show high potency.

Results

CA inhibition. Inhibition data against isozymes I, II, IV and IX withcompounds 71-91 reported here are shown in Table 3.

TABLE 3 Inhibition of isozymes hCA I, hCA II, bCA IV and hCA IX with thepyridinium salts 71-91.

K_(I)*(nM) hCA R¹ R² R³ R⁴ R⁵ hCA I^(a) hCA II^(a) bCA IV^(b) IX^(c) 71Me H Me H Me 4 0.26 2.1 3 72 i-Pr H Me H Me 4 0.39 3.0 5 73 i-Pr H Me Hi-Pr 7 1.54 4.7 16 74 t-Bu H Me H t-Bu 11 3.13 9.4 34 75 Me H Ph H Me 30.20 2.0 6 76 Et H Ph H Et 4 0.21 2.3 9 77 n-Pr H Ph H n-Pr 9 3.45 8.135 78 n-Bu H Ph H n-Bu 10 4.62 10.3 40 79 i-Pr H Ph H i-Pr 5 1.61 4.1 3080 Me H Ph H Ph 4 1.21 3.0 24 81 Et H Ph H Ph 5 1.14 3.8 29 82 n-Pr H PhH Ph 8 3.90 6.0 40 83 i-Pr H Ph H Ph 6 3.74 4.5 32 84 n-Bu H Ph H Ph 84.95 8.4 45 85 t-Bu H Ph H Ph 12 4.11 7.0 43 86 Ph H Me H Ph 6 4.78 5.812 87 Ph H Ph H Ph 5 5.96 5.6 12 88 Ph H H H Ph 5 4.93 5.4 16 89 Me MeMe H Me 3 0.30 2.4 5 90 Me Me Ph H Me 3 1.24 5.2 15 91 Me R³, R⁵ = Me 31.37 4.6 12 (CH₂)₉; R⁴ = Me aminobenzolamide 6 2.04 5.1 38 acetazolamide250 12 70 25 methazolamide 50 14 36 27 dichlorophenamide 1200 38 380 50indisulam 30 15 65 24 ^(a)Human (cloned) isozymes, esterase assay method[76]. ^(b)From bovine lung microsomes, esterase assay method [76].^(c)Catalytic domain of the human, cloned isozyme, CO₂ hydrase assaymethod [44]. *Errors in the range of ± 10% of the reported value, fromthree different determinations.

Ex vivo penetration through red blood cells. Levels of sulfonamides inred blood cells after incubation of human erythrocytes with millimolarsolutions of inhibitor for 30-60 min (both classical as well aspositively-charged sulfonamides were used in such experiments) are shownin Table 4 [4, 12, 36, 45, 53, 54, 58, 59, 84, 116, 118].

TABLE 4 Levels of sulfonamide CA inhibitors (μM) in red blood cells at30 and 60 min, after exposure of 10 mL of blood to solutions ofsulfonamide (2 mM sulfonamide in 5 mM Tris buffer, pH 7.4). Theconcentrations of sulfonamide has been determined by three methods:HPLC; electronic spectroscopy (ES) and the enzymatic method (EI) - seeExperimental for details. [sulfonamide], μM* t = 30 min t = 60 minInhibitor HPLC^(a) ES^(b) EI^(c) HPLC^(a) ES^(b) EI^(c) AAZ 136 139 140160 167 163 MZA 170 169 165 168 168 167 Benzolamide 110 108 112 148 146149 Aminobenzolamide 125 127 122 154 156 158 71 0.3 0.5 0.5 0.4 0.5 0.376 1.0 1.1 1.0 1.1 1.2 1.1 89 0.3 0.2 0.5 0.3 0.6 0.4 91 0.4 0.3 0.5 0.30.6 0.5 *Standard error (from 3 determinations) <5% by: ^(a)the HPLCmethod [36]; ^(b)the electronic spectroscopic method [4]; ^(c)theenzymatic method [76].

The new compounds reported in the present work were characterized bystandard chemical and physical methods (elemental analysis, within ±0.4%of the theoretical values; IR and NMR spectroscopy) that confirmed theirstructure (see Materials and Methods and Table 5 below for details) andwere assayed for the inhibition of isozymes hCA I, hCA II, bCA IV andhCA IX.

TABLE 5 Elemental analysis data for the compounds described in Example 3Elemental analysis data (calc./found) No Formula % C % H % N 71C₁₆H₁₈N₅O₄S₃ ⁺ClO₄ ⁻ 35.59/35.32 3.36/3.62 12.97/12.93 72 C₁₈H₂₂N₅O₄S₃⁺ClO₄ ⁻ 38.06/37.95 3.90/4.16 12.33/12.18 73 C₂₀H₂₆N₅O₄S₃ ⁺ClO₄ ⁻40.30/39.99 4.40/4.54 11.75/11.63 74 C₂₂H₃₀N₅O₄S₃ ⁺ClO₄ ⁻ 42.34/42.564.84/4.76 11.22/11.03 75 C₂₁H₂₀N₅O₄S₃ ⁺ClO₄ ⁻ 41.89/42.02 3.35/3.0311.63/11.48 76 C₂₃H₂₄N₅O₄S₃ ⁺ClO₄ ⁻ 43.84/43.88 3.84/3.62 11.11/10.95 77C₂₅H₂₈N₅O₄S₃ ⁺ClO₄ ⁻ 45.62/45.60 4.29/4.36 10.64/10.50 78 C₂₇H₃₂N₅O₄S₃⁺ClO₄ ⁻ 47.26/47.45 4.70/4.89 10.21/10.14 79 C₂₅H₂₈N₅O₄S₃ ⁺ClO₄ ⁻45.62/45.49 4.29/4.18 10.64/10.61 80 C₂₆H₂₂N₅O₄S₃ ⁺ClO₄ ⁻ 47.02/46.793.34/3.33 10.55/10.23 81 C₂₇H₂₄N₅O₄S₃ ⁺ClO₄ ⁻ 47.82/47.73 3.57/3.7310.33/10.40 82 C₂₈H₂₆N₅O₄S₃ ⁺ClO₄ ⁻ 48.59/48.83 3.79/3.91 10.12/10.24 83C₂₈H₂₆N₅O₄S₃ ⁺ClO₄ ⁻ 48.59/48.27 3.79/3.82 10.12/10.05 84 C₂₉H₂₈N₅O₄S₃⁺ClO₄ ⁻ 49.32/49.59 4.00/4.23 9.92/9.67 85 C₂₉H₂₈N₅O₄S₃ ⁺ClO₄ ⁻49.32/49.16 4.00/3.94 9.92/9.71 86 C₂₆H₂₂N₅O₄S₃ ⁺ClO₄ ⁻ 47.02/47.253.34/3.18 10.55/10.46 87 C₃₁H₂₄N₅O₄S₃ ⁺ClO₄ ⁻ 51.27/51.50 3.33/3.609.64/9.67 88 C₂₅H₂₀N₅O₄S₃ ⁺ClO₄ ⁻ 46.19/46.28 3.10/2.95 10.77/10.67 89C₁₇H₂₀N₅O₄S₃ ⁺ClO₄ ⁻ 36.86/36.72 3.64/3.53 12.64/12.45 90 C₂₂H₂₂N₅O₄S₃⁺ClO₄ ⁻ 42.89/42.70 3.60/3.84 11.37/11.15 91 C₂₄H₃₂N₅O₄S₃ ⁺ClO₄ ⁻44.34/44.57 4.96/4.99 10.77/10.51Conclusions

We report here a general approach for the preparation ofpositively-charged, membrane-impermeant sulfonamide CA inhibitors withhigh affinity for the cytosolic isozymes CA I and CA II, as well as forthe membrane-bound ones CA IV and CA IX. They were obtained by attachingsubstituted-pyridinium moieties to aminobenzolamide, a very potent CAinhibitor itself. Ex vivo studies showed the new class of inhibitorsreported here to discriminate for the membrane-bound versus thecytosolic isozymes. Correlated with the low nanomolar affinity of someof these compounds for the tumor-associated isozyme CA IX, this reportconstitutes the basis of selectively inhibiting only the target,tumor-associated CA IX in vivo, whereas the cytosolic isozymes wouldremain unaffected.

Characterization of Compounds 71-91 (For Preparation, see Materials andMethods Section)

1-N-[5-Sulfamoyl-1,3,4-thiadiazol-2-yl-(aminosulfonyl-4-phenyl)]-2,4,6-trimethyl-pyridiniumperchlorate 71: white crystals, mp >300° C.; IR (KBr), cm⁻¹ (bands initalics are due to the anion): 595, 625, 664, 787, 803, 884, 915, 1100,1150, 1190, 1200, 1285, 1360, 1495, 1604, 3065; ¹H-NMR (D₂O), δ, ppm:3.08 (s, 6H, 2,6-Me₂); 3.11 (s, 3H, 4-Me), 7.30-8.06 (m, AA′BB′, 4H, ArHfrom phenylene); 9.05 (s, 2H, ArH, 3,5-H from pyridinium); in thissolvent the sulfonamido protons are not seen, being in fast exchangewith the solvent. Anal C₁₆H₁₈N₅O₄S₃ ⁺ClO₄ ⁻ (C, H, N).

1-N-[5-Sulfamoyl-1,3,4-thiadiazol-2-yl-(aminosulfonyl-4-phenyl)]-2-iso-propyl-4,6-dimethylpyridiniumperchlorate 72, colorless crystals, mp 29o-1° C.; IR (KBr), cm⁻¹: 625,680, 720, 1100, 1165, 1330, 1640, 3020, 3235; ¹H-NMR (TFA), δ, ppm: 1.50(d, 6H, 2Me from i-Pr); 2.80 (s, 3H, 6-Me); 2.90 (s, 3H, 4-Me); 3.49(heptet, 1H, CH from i-Pr); 7.25-8.43 (m, AA′BB′, 4H, ArH from1,4-phenylene); 7.98 (s, 2H, ArH, 3,5-H from pyridinium). AnalC₁₈H₂₂N₅O₄S₃ ⁺ ClO₄ ⁻ (C, H, N).

1-N-[5-Sulfamoyl-1,3,4-thiadiazol-2-yl-(aminosulfonyl-4-phenyl)]-2,6-di-iso-propyl-4-methylpyridiniumperchlorate 73, tan crystals, mp 278-9° C.; IR (KBr), cm⁻¹: 625, 685,820, 1100, 1165, 1340, 1635, 3030, 3250; ¹H-NMR (TFA), δ, ppm: 1.51 (d,12H, 4Me from 2 i-Pr); 2.83 (s, 3H, 4-Me); 3.42 (heptet, 2H, 2CH from 2i-Pr); 7.31-8.51 (m, AA′BB′, 4H, ArH from 1,4-phenylene); 8.05 (s, 2H,ArH, 3,5-H from pyridinium). Anal C₂₀H₂₆N₅O₄S₃ ⁺ ClO₄ ⁻ (C, H, N).

1-N-[5-Sulfamoyl-1,3,4-thiadiazol-2-yl-(aminosulfonyl-4-phenyl)]-2,6-dimethyl-4-phenylpyridiniumperchlorate 75, white crystals, mp >300° C.; IR (KBr), cm⁻¹: 625, 690,770, 1100, 1170, 1330, 1635, 3030, 3260, 3330; ¹H-NMR (TFA), δ, ppm:2.62 (s, 6H, 2,6-(Me)₂); 8.10-9.12 (m, H, ArH from 1,4-phenylene,pyridinium and 4-Ph). Anal C₂₁H₂₀N₅O₄S₃ ⁺ ClO₄ ⁻ (C, H, N).

1-N-[5-Sulfamoyl-1,3,4-thiadiazol-2-yl-(aminosulfonyl-4-phenyl)]-2,6-diethyl-4-phenylpyridiniumperchlorate 76, tan crystals, mp 267-8° C.; IR (KBr), cm⁻¹: 625, 695,765, 1100, 1180, 1340, 1630, 3040, 3270, 3360; ¹H-NMR (TFA), δ, ppm:1.43 (t, 6H, 2 Me from ethyl); 2.82 (q, 4H, 2 CH₂ from Et); 7.68-8.87(m, 11H, ArH from 1,4-phenylene, pyridinium and 4-Ph). Anal C₂₃H₂₄N₅O₄S₃⁺ ClO₄ ⁻ (C, H, N).

1-N-[5-Sulfamoyl-1,3,4-thiadiazol-2-yl-(aminosulfonyl-4-phenyl)]-2,6-di-n-propyl-4-phenylpyridiniumperchlorate 77, colorless crystals, mp 235-7° C.; IR (KBr), cm⁻¹: 625,695, 770, 1100, 1180, 1340, 1630, 3050, 3220, 3315; ¹H-NMR (TFA), δ,ppm: 1.06 (t, 6H, 2 Me from propyl); 1.73 (sextet, 4H, 2CH₂ (β) fromn-Pr); 2.84 (t, 4H, 2 CH₂ (α) from n-Pr); 7.55-8.71 (m, 11H, ArH from1,4-phenylene, pyridinium and 4-Ph). Anal C₂₅H₂₈N₅O₄S₃ ⁺ ClO₄ ⁻ (C, H,N).

1-N-[5-Sulfamoyl-1,3,4-thiadiazol-2-yl-(aminosulfonyl-4-phenyl)]-2,6-di-isopropyl-4-phenylpyridiniumperchlorate 79, white crystals, mp 278-9° C.; IR (KBr), cm⁻¹: 625, 690,765, 1100, 1180, 1340, 1625, 3040, 3270, 3315; ¹H-NMR (TFA), δ, ppm:1.45 (d, 12H, 4 Me from i-Pr); 2.95 (heptet, 2H, 2 CH from i-Pr);7.92-8.97 (m, 11H, ArH from 1,4-phenylene, pyridinium and 4-Ph). AnalC₂₅H₂₈N₅O₄S₃ ⁺ ClO₄ ⁻ (C, H, N).

1-N-[5-Sulfamoyl-1,3,4-thiadiazol-2-yl-(aminosulfonyl-4-phenyl)]-2-methyl-4,6-diphenylpyridiniumperchlorate 80, white crystals, mp 298-99° C.; IR (KBr), cm⁻¹: 625, 710,770, 1100, 1170, 1345, 1625, 3040, 3245, 3350; ¹H-NMR (TFA), δ, ppm:2.75 (s, 3H, 2-Me); 7.53-8.70 (m, 16H, ArH from 1,4-phenylene,pyridinium and 4,6-Ph₂). Anal C₂₆H₂₂N₅O₄S₃ ⁺ ClO₄ ⁻ (C, H, N).

1-N-[5-Sulfamoyl-1,3,4-thiadiazol-2-yl-(aminosulfonyl-4-phenyl)]-2-ethyl-4,6-diphenylpyridiniumperchlorate 81, white crystals, mp 254-5° C.; IR (KBr), cm⁻¹: 625, 700,770, 1100, 1180, 1340, 1620, 3040, 3250, 3350; ¹H-NMR (TFA), δ, ppm:1.52 (t, 3H, Me from ethyl); 2.97 (q, 2H, CH₂); 7.40-8.57 (m, 16H, ArHfrom 1,4-phenylene, pyridinium and 4,6-Ph₂). Anal C₂₇H₂₄N₅O₄S₃ ⁺ ClO₄ ⁻(C, H, N).

1-N-[5-Sulfamoyl-1,3,4-thiadiazol-2-yl-(aminosulfonyl-4-phenyl)]-2-n-propyl-4,6-diphenylpyridiniumperchlorate 82, white crystals, mp 214-5° C.; IR (KBr), cm⁻¹: 625, 700,770, 1100, 1180, 1340, 1620, 3030, 3270, 3350; ¹H-NMR (TFA), δ, ppm:1.03 (t, 3H, Me from propyl); 1.95 (sextet, 2H, β-CH₂ from n-Pr); 2.88(t, 2H, α-CH₂ from n-Pr); 7.39-8.55 (m, 16H, ArH from 1,4-phenylene,pyridinium and 4,6-Ph₂). Anal C₂₈H₂₆N₅O₄S₃ ⁺ ClO₄ ⁻ (C, H, N).

1-N-[5-Sulfamoyl-1,3,4-thiadiazol-2-yl-(aminosulfonyl-4-phenyl)]-2-iso-propyl-4,6-diphenylpyridiniumperchlorate 83, white crystals, mp 186-8° C.; IR (KBr), cm⁻¹: 625, 700,770, 1100, 1170, 1340, 1620, 3040, 3250, 3360; ¹H-NMR (TFA), δ, ppm:1.51 (d, 6H, 2 Me from i-propyl); 2.50-3.27 (m, 1H, CH from i-Pr);7.32-8.54 (m, 16H, ArH from 1,4-phenylene, pyridinium and 4,6-Ph₂). AnalC₂₈H₂₆N₅O₄S₃ ⁺ ClO₄ ⁻ (C, H, N).

1-N-[5-Sulfamoyl-1,3,4-thiadiazol-2-yl-(aminosulfonyl-4-phenyl)]-2-n-butyl-4,6-diphenylpyridiniumperchlorate 84, white crystals, mp 241-3° C.; IR (KBr), cm⁻¹: 625, 710,770, 1100, 1180, 1335, 1625, 3040, 3260, 3345; ¹H-NMR (TFA), δ, ppm:0.93 (t, 3H, Me from butyl); 1.12-2.14 (m, 4H, CH₃—CH₂—CH₂—CH₂ fromn-Bu); 2.96 (t, 2H, α-CH₂ from n-Bu); 7.21-8.50 (m, 16H, ArH from1,4-phenylene, pyridinium and 4,6-Ph₂). Anal C₂₉H₂₈N₅O₄S₃ ⁺ ClO₄ ⁻ (C,H, N).

1-N-[5-Sulfamoyl-1,3,4-thiadiazol-2-yl-(aminosulfonyl-4-phenyl)]-2-tert-butyl-4,6-diphenylpyridiniumperchlorate 85, white crystals, mp 203-5° C.; IR (KBr), cm⁻¹: 625, 705,765, 1100, 1160, 1310, 1620, 3060, 3270; ¹H-NMR (TFA), δ, ppm: 1.91 (s,9H, t-Bu); 6.80-8.74 (m, 16H, ArH from 1,4-phenylene, 4,6-Ph₂ and 3,5-Hfrom pyridinium). Anal C₂₉H₂₈N₅O₄S₃ ⁺ ClO₄ ⁻ (C, H, N).

1-N-[5-Sulfamoyl-1,3,4-thiadiazol-2-yl-(aminosulfonyl-4-phenyl)]-2,4,6-triphenhyl-pyridiniumperchlorate 87: pale yellow crystals, mp >300° C.; IR (KBr), cm⁻¹ (bandsin italics are due to the anion): 625, 635, 703, 785, 896, 1100, 1150,1204, 1355, 1410, 1520, 1600, 3065; ¹H-NMR (D₂O), δ, ppm: 7.50-8.60 (m,19H, ArH, 3Ph+C₆H₄); 9.27 (s, 2H, ArH, 3,5-H from pyridinium); in thissolvent the sulfonamido protons are not seen, being in fast exchangewith the solvent. Anal C₃₁H₂₄N₅O₄S₃ ⁺ ClO₄ ⁻ (C, H, N).

1-N-[5-Sulfamoyl-1,3,4-thiadiazol-2-yl-(aminosulfonyl-4-phenyl)]-2,6-diphenylpyridiniumperchlorate 88, yellow crystals, mp 218-20° C.; IR (KBr), cm⁻¹: 625,705, 765, 1100, 1160, 1335, 1615, 3050, 3260; ¹H-NMR (TFA), δ, ppm:6.75-8.43 (m, 17H, ArH from 1,4-phenylene, 2,6-Ph₂ and 3,4,5-H frompyridinium). Anal C₂₅H₂₀N₅O₄S₃ ⁺ ClO₄ ⁻ (C, H, N).

1-N-[5-Sulfamoyl-1,3,4-thiadiazol-2-yl-(aminosulfonyl-4-phenyl)]-2,3,4,6-tetramethylpyridiniumperchlorate 89, tan crystals, mp >300° C.; IR (KBr), cm⁻¹: 625, 800,1100, 1165, 1330, 1630, 3030, 3305; ¹H-NMR (TFA), δ, ppm: 2.62 (s, 3H,4-Me); 2.74 (s, 3H, 3-Me); 2.88 (s, 6H, 2,6-(Me)₂); 7.21-8.50 (m,AA′BB′, 4H, ArH from 1,4-phenylene); 7.93 (s, 1H, ArH, 5-H frompyridinium). Anal C₁₇H₂₀N₅O₄S₃ ⁺ ClO₄ ⁻ (C, H, N).

Example 4 Ectopic Expression of CA IX Leads to Increased Acidificationof Extracellular pH in Hypoxia

Expression of CA IX in tumor cells is induced by hypoxia simultaneouslywith various components of anaerobic metabolism and acid extrusionpathways. Such simultaneous induction complicates the determination ofthe contribution of CA IX to the overall change in pHe. Therefore, theinventors used MDCK immortalized canine kidney epithelial cells that donot express endogenous CA IX, but were stably transfected to express thehuman CA IX protein in a constitutive manner. Levels of CA IX in MDCK-CAIX transfectants were comparable between the hypoxic cells maintainedfor 48 hours in 2% O₂ and the normoxic cells incubated in 21% O₂ (datanot shown). Immunofluorescence analysis indicated that CA IX waspredominantly localized at the cell surface, although the membranestaining in hypoxic cells was less pronounced due to hypoxia-inducedperturbation of intercellular contacts [103]. Hypoxic incubation led tothe expected extracellular acidification in the CA IX-positive as wellas CA IX-negative cell cultures when compared to their normoxiccounterparts (FIG. 7A). However, upon mutual comparison of the hypoxiccells it was evident that pHe was significantly decreased in the cellscontaining CA IX. Taking into account a steady, hypoxia-independentlevel of CA IX in MDCK-CA IX cells, that finding indicated that hypoxiaactivated the catalytic performance of CA IX, which resulted in enhancedpHe acidification.

To exclude the possibility that hypoxia-induced acidification was causedby an increased production of lactic acid, the inventors determined thecorresponding lactate concentrations in the media from both CAIX-negative and CA IX-positive transfectants (FIG. 7B). In accord withthe literature, production of lactic acid was significantly higher inthe cells maintained in hypoxia than in the normoxic cells. However,there were practically no differences between the lactate production incultures of CA IX-positive and CA IX-negative cells, suggesting that theexcessive pHe decrease observed in hypoxia could be explained by theactivation of CA IX.

Example 5 Sulfonamides Inhibit CA IX-Mediated Acidification of pHe andBind to Hypoxic MDCK-CA IX Cells

The three representative CA IX-selective inhibitors shown in FIG. 8Awere tested in accordance with the concepts of the subject invention.Compound 6 is a strong inhibitor of CA IX, whereas it is less efficientagainst the widely distributed cytoplasmic CA II and the plasmamembrane-anchored CA IV [114], Compound 39 is practicallymembrane-impermeable [81] and Compound 92 [FITC derivative ofhomosulfanilamide (Compound 5)] has a big moiety favoring itsinteraction with the CA IX active site, which is assumed to form alarger cavity than in CA II [135]. All three sulfonamides were able toreduce the extracellular acidification of MDCK-CA IX cells in hypoxiaand their effect on the normoxic pHe was negligible (FIG. 8B). Moreover,in fluorescence analysis (treated MDCK-CA IX cells incubated in normoxiaor hypoxia for 48 hours), FITC-labeled Compound 92 was detected only inhypoxic MDCK-CA IX cells, but was absent from their normoxiccounterparts and from the mock-transfected controls (data not shown).Cytoplasmic accumulation of Compound 92 was possibly related to ahypoxia-induced internalization of CA IX described earlier [103]. Lackof the fluorescence signal in CA IX-negative MDCK cells confirmed theselectivity of the inhibitor, which did not bind to other potentiallypresent CA isoforms and indicated that only the hypoxic MDCK-CA IX cellscontain the catalytically active CA IX with the enzyme center accessibleto inhibitor.

Example 6 Intact CA IX Catalytic Domain is Required for theExtracellular Acidification in Hypoxia

In addition to the enzyme domain (CA), the extracellular part of CA IXcontains an N-terminal proteoglycan-related region (PG) that is absentfrom the other CAs and seems implicated in cell adhesion [146]. Toexamine involvement of those CA IX domains in pHe control, the inventorsproduced deletion variants of CA IX, in which either the PG region (ΔPG)or a large portion of the CA domain (ΔCA) was removed [FIG. 9A].Immunofluorescence analysis using two MAbs, namely the PG-specific M75for ΔCA and CA-specific V/10 for ΔPG, has shown that both deletedproteins were transported to the plasma membrane (data not shown). Themutants were expressed at levels comparable with the wild-type CA IX, asanalyzed by immunoblotting of ΔCA and ΔPG proteins under both reducingand non-reducing conditions for their molecular weight and a capacity toform oligomers (data not shown). Interestingly, ΔCA was unable to formoligomers possibly due to the absence of two out of four cysteines (C174and C336) required for the proper S—S bonding. As judged from themolecular weights, ΔPG mutant appeared to assemble into dimeric andtetrameric complexes, rather then into trimers.

Elimination of a large part of the CA domain perturbed the acidificationcapacity of CA IX, whereas removal of the PG region had no such effect(FIG. 9B). That differential behavior could be reasonably assigned tothe absence versus presence of the catalytic activity of CA IX, becausethe cells expressing these variants produced similar levels of lacticacid (FIG. 9B). It also indicates that the CA domain is both necessaryand sufficient for the enzyme activity, and that the PG and CA portionsof CA IX can be functionally separated, although they may stillcooperate in response to diverse physiological factors. Based on theknowledge that the extracellular acidosis interferes with the celladhesion, the enzyme activity carried out by the CA domain mightinfluence the adhesion-related properties of PG region and vice versa.Indeed, CA IX was shown to destabilize E cadherin-mediated intercellularadhesion in transfected MDCK cells, which was particularly dramatic inthe hypoxic monolayer [103] in conditions accompanied by CA IX-mediatedextracellular acidosis described herein.

Example 7 FITC-Labeled Compound 92 Sulfonamide Binds to and IncreasespHe of Hypoxic Tumor Cells

To see whether the phenomenon of CA IX-mediated acidification isapplicable to tumor cells with endogenous CA IX, the effect ofFITC-labeled Compound 92 sulfonamide [FITC derivative ofhomosulfanilamide, Compound 5] on the pHe of the cervical carcinomacells HeLa and SiHa, respectively, was examined. Under hypoxia, tumorcells coordinately express elevated levels of multiple HIF-1 targets,including CA IX [139]. In addition, activity of many components of thehypoxic pathway and related pH control mechanisms, such as ion transportacross the plasma membrane, are abnormally increased in order tomaintain the neutral intracellular pH [86]. This explains theconsiderably decreased pHe of the hypoxic versus normoxic HeLa and SiHacells (FIG. 10). The acidosis was partially reduced by Compound 92inhibitor, in support of the idea that activation of CA IX is just oneof many consequences of hypoxia. Moreover, FITC-labeled Compound 92accumulated in the hypoxic HeLa and SiHa cells that contained elevatedlevels of CA IX, but not in the normoxic cells with a diminished CA IXexpression. [HeLa and SiHa cells plated on coverslips were treated withFITC-labeled Compound 92 sulfonamide during 48 hour incubation innormoxia and hypoxia, washed with PBS and inspected under thefluorescence microscope (data not shown). CA IX expression levels weremeasured by immunoblotting analysis with M75 MAb.] As indicated by CAIX's ability to bind Compound 92 and mediate its accumulation inhypoxia, CA IX expressed in the hypoxic tumor cells was catalyticallyactive.

Example 8 Expression of ΔCA Mutant in HeLa Cells Reduces pHeAcidification in Hypoxia

Based on the assumption that the enzyme-dead ΔCA mutant could abolishthe function of the endogenous CA IX, the inventors generated HeLa-ΔCAtransfectants. As determined by immunofluorescence and immunoblottinganalysis (using V/10 MAb to detect endogenous CA IX protein or M75 MAbto visualize both CA IX and ΔCA mutant), the HeLa-ΔCA cells containedΔCA but not CA IX under normoxia, expressed both proteins under hypoxia,and under non-reducing conditions exhibited an atypical band presumablycorresponding to mixed oligomers composed of both CA IX and ΔCA (datanot shown). No significant differences in pHe were observed between thenormoxic HeLa-mock and HeLa-ΔCA cells. On the other hand, HeLa-ΔCAtransfectants treated by hypoxia produced less acidic medium than thecontrol HeLa-mock cells (FIG. 11), suggesting that the inactive ΔCAdeletion variant interfered with the activity of the wild-type protein,and further supporting the role of CA IX. Altogether, the data stronglyimplies that the acidification of the extracellular pH in hypoxic tumorcells does involve CA activity, and that CA IX directly participates inthe phenomenon.

DISCUSSION

In the context of the experimental results described herein that placeCA IX among the direct contributors to the hypoxic microenvironment, itis tempting to propose possible means of CA IX's action. MN/CA IX isconsidered to participate in this phenomenon by catalyzing hydration ofcarbon dioxide to generate bicarbonate ions that are then transportedinto cell interior and protons that acidify extracellular pH. There aresome indications given by the data obtained with the physiologicallyrelevant CA isoforms II and IV that physically interact with anionexchangers (AE) to form a metabolon that facilitates bicarbonatetransport in differentiated cells [140, 141]. It seems plausible that CAIX could work as an extracellular component of the similar metabolon intumor cells. Assembly and/or activation of such metabolon would beespecially meaningful in low oxygen conditions, because a highlyefficient transport of bicarbonate is required particularly in hypoxiccells for the buffering of intracellular pH and biosynthetic reactions.According to such a model, enhanced conversion of CO₂ to bicarbonate bythe hypoxia-activated CA IX would be coupled with the increasedproduction of extracellular protons contributing to acidosis. Dataobtained as disclosed herein fit well with such a proposal. Furthersupportive hints come from the studies of von Hippel-Lindau tumorsuppressor protein (pVHL), the main negative regulator of HIF-1, whichcan down-regulate CA IX (obviously as a direct HIF-1 target) and canalso reduce the transport activity of AEs [128, 131].

Downstream effects of CA IX can be at least partially anticipated on thebasis of the known connections between the acidic pHe and certainfeatures of the tumor phenotype [86, 124, 125, 126, 130, 132]. Moreover,as a part of the hypoxic acidification machinery, CA IX might facilitatea nucleolar sequestration of pVHL and activation of HIF, which is arecently described pH-dependent mechanism proposed to serve a protectiverole in reoxygenated cells [Mekhail et al., 2004]. In such case,HIF-mediated increase in the level and activity of CA IX resulting inenhanced acidification might create a feedback loop leading to aprolonged HIF activation, which is certainly an attractive possibilityrequiring experimental proof.

In conclusion, the instant disclosure provides the first direct evidencefor the role of CA IX in acidification of the extracellular pH. Thefindings of the inventors significantly improve the view of CA IX as amolecule, whose levels and catalytic activity are regulated by theoxygen availability, and open new possibilities for its betterunderstanding and clinical exploitation. Inhibition of the MN/CA IXcatalytic activity resulting in reduced extracellular acidification mayhave direct anticancer effects or may modulate efficiency of thoseconventional chemotherapeutic drugs whose uptake is pH-dependent.

Budapest Treaty Deposits

The hybridoma VU-M75 was deposited on Sep. 17, 1992 with the AmericanType Culture Collection (ATCC) now at 10810 University Blvd., Manassus,Va. 20110-2209 (USA) and assigned Accession No. HB 11128. The depositwas made under the provisions of the Budapest Treaty on theInternational Recognition of Deposited Microorganisms for the Purposesof Patent Procedure and Regulations thereunder (Budapest Treaty).Maintenance of a viable culture is assured for thirty years from thedate of deposit. The hybridoma will be made available by the ATCC underthe terms of the Budapest Treaty, and subject to an agreement betweenthe Applicants and the ATCC which assures unrestricted availability ofthe deposited hybridoma to the public upon the granting of a patent fromthe instant application. Availability of the deposited strain is not tobe construed as a license to practice the invention in contravention ofthe rights granted under the authority of any Government in accordancewith its patent laws.

Similarly, the hybridoma cell line V/10-VU which produces the V/10monoclonal antibodies was deposited on Feb. 19, 2003 under the BudapestTreaty at the International Depository Authority (IDA) of the BelgianCoordinated Collections of Microorganisms (BCCM) at the Laboratoriumvoor Moleculaire Biologie-Plasmidencollectie (LMBP) at the UniverseitGent, K.L. Ledeganckstraat 35, is B-9000 Gent, Belgium [BCCM/LMBP] underthe Accession No. LMBP 6009CB.

The description of the foregoing embodiments of the invention have beenpresented for purposes of illustration and description. They are notintended to be exhaustive or to limit the invention to the precise formdisclosed, and obviously many modifications and variations are possiblein light of the above teachings. The embodiments were chosen anddescribed in order to explain the principles of the invention and itspractical application to enable thereby others skilled in the art toutilize the invention in various embodiments and with variousmodifications as are suited to the particular use contemplated.

All references cited herein are hereby incorporated by reference.

1. A method of selectively imaging activated MN/CA IX expressed inhypoxic tissues in a patient, comprising: a) administering to saidpatient a specific inhibitor of activated MN/CA IX, wherein saidspecific inhibitor of activated MN/CA IX is an MN/CA IX-specificantibody that binds the activated form of MN/CA IX's CA domain and notthe inactive form of MN/CA IX's CA domain, and said antibody is linkedto an imaging agent; and b) if activated MN/CA IX is present in saidhypoxic tissues, detecting the binding of said MN/CA IX-specificantibody, wherein binding of said MN/CA IX-specific antibody indicatesthe presence of activated MN/CA IX in said tissues.
 2. Adiagnostic/prognostic method for a preneoplastic/neoplastic diseaseassociated with abnormal MN/CA IX expression, comprising determiningwhether MN/CA IX is activated in a vertebrate sample, comprising: a)contacting said sample with a specific inhibitor of the activated formof MN/CA IX's CA domain, wherein said specific inhibitor of theactivated form of MN/CA IX's CA domain is an MN/CA IX-specific antibodythat binds the activated form of MN/CA IX's CA domain and not theinactive form of MN/CA IX's CA domain, and b) detecting or detecting andquantifying binding of said specific inhibitor of the activated form ofMN/CA IX's CA domain in said sample; wherein binding of said inhibitorto MN/CA IX indicates that said bound MN/CA IX in said sample isactivated.
 3. The method of claim 2 wherein said activated MN/CA IX ishypoxia-activated.
 4. The method of claim 2, wherein said MN/CAIX-specific antibody, that is a specific inhibitor of the activated formof MN/CA IX's CA domain, is conjugated to a label or a visualizingmeans, wherein said detecting or detecting and quantifying bindingcomprises detecting or detecting and quantifying said label or saidvisualizing means on cells in said sample, and wherein said detecting orsaid detecting and quantifying at a level above that for a controlsample is indicative of hypoxic precancerous or cancerous cells thatabnormally express activated MN/CA IX in said sample.
 5. The method ofclaim 4, wherein said label is fluorescein isothiocyanate.
 6. The methodof claim 4, further comprising detecting the binding of a secondantibody that specifically binds to a MN/CA IX domain other than thecarbonic anhydrase domain.
 7. The method of claim 4, wherein said methodis used as an aid in selection of patient therapy.
 8. The method ofclaim 7, wherein said binding to activated MN/CA IX is detectable at alevel above that for a control sample, and said method is used in thedecision to use hypoxia-selective therapy.
 9. The method of claim 8,wherein said hypoxia-selective therapy comprises the use of drugs thatare toxic only under hypoxic conditions.
 10. The method of claim 9,wherein said hypoxia-selective therapy comprises the use of tirapazamineor AQ4N.
 11. The method of claim 7, wherein said binding to activatedMN/CA IX is not detectable at a level above that for a control sample,and said method is used in the decision to use radiotherapy ornon-hypoxia-selective chemotherapy.
 12. A method that is diagnostic, ordiagnostic and prognostic for precancer or cancer associated withoverexpression of activated MN/CA IX comprising contacting a mammaliansample with a specific inhibitor of activated MN/CA IX, wherein saidspecific inhibitor of activated MN/CA IX is an MN/CA IX-specificantibody that specifically binds to the activated form of MN/CA IX's CAdomain and does not bind the inactive form of MN/CA IX'S CA domain, saidantibody conjugated to a label or a visualizing means, and detecting, ordetecting and quantifying binding of said MN/CA IX-specific antibody tocells in said sample by detecting, or detecting and quantifying saidlabel or said visualizing means on cells in said sample, wherein saiddetection, or said detection and quantitation at a level above that fora control sample is indicative of precancerous or cancerous cells thatoverexpress activated MN/CA IX in said sample.
 13. The method of claim12 wherein MN/CA IX activated by hypoxic conditions is detected, ordetected and quantitated, and the mammal from whom the sample was takenis considered to have a poor prognosis, and decisions on treatment forsaid mammal are made in view of the presence of said hypoxic conditions.